Polypeptides having L-asparaginase activity

ABSTRACT

Disclosed are polypeptides which originate from mammal, having L-asparaginase activity. The polypeptides are easily prepared by applying recombinant DNA techniques to DNAs encoding the polypeptides and they exert satisfactory effects in the treatment and/or the prevention for diseases caused by tumor cells dependent on L-asparagine, and cause no substantial serious side effects even when administered to humans in relatively-high dose.

This is a continuation of copending parent application Ser. No.08/869,927, filed Jun. 5. 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to L-asparagine amidohydrolytic enzymes,more particularly, to polypeptides which originate from mammal, havingL-asparaginase activity.

2. Description of the Prior Art

L-Asparaginase (EC 3.5.1.1) is an enzyme which catalyzes the hydrolyticreaction of L-asparagine into L-aspartic acid and ammonia. The studieson the antitumor activity of L-asparaginase started from the followingreports: J. G. Kidd et al. described the inhibitory action of guinea pigsera on cells of lymphomas in “The Journal of Experimental Medicine”,Vol.98, pp.565-582 (1953), and J. D. Broome et al. evidenced in“Nature”, Vol.191, pp.1,114-1,115 (1961), that the L-asparaginaseactivity of the guinea pig sera was responsible for the inhibitoryaction. It is now understood that the inhibitory action is caused by thelack of L-asparagine, an essential nutrient to proliferate and survivefor some tumor cells which defect L-asparagine synthetase activity, suchas acute lymphocytic leukemia, but not for normal cells. The hydrolysisof L-asparagine by L-asparaginase in patients with such tumor cellsinduces selective death of the tumor cells, resulting in the treatmentof malignant tumors.

L-Asparaginase has been studied energetically for its actual use as anantitumor agent, and one derived from Escherichia coli is now in use asa therapeutic agent for leukemia and lymphoma. However, L-asparaginasefrom Escherichia coli is merely an external protein for human, andrepetitive administration of conventional compositions with suchL-asparaginase may cause serious side effects such as anaphylaxis shock,urticaria, edema, wheeze and dyspnea. These compositions are inevitablyrestricted with respect to administration dose and frequency. Therefore,some proposals to reduce or even diminish such side effects have beengiven.

As a first proposal, Japanese Patent Kokai No.119,082/79 discloses achemically modified L-asparaginase from Escherichia coli, in which atleast 65% amino acids are blocked with 2-O- substituted polyethyleneglycol-4,6-dichloro-S-triazine. As a second proposal, humanL-asparaginases are disclosed in Japanese Patent Kokai Nos.320,684/92and 19,018/80, where the L-asparaginases are respectively obtained fromcultures of human cell lines and human urine. While the first proposalhas an advantage of that the L-asparaginase from Escherichia coli iseasily obtainable on an industrial scale, it has a disadvantage of thatthe modifying reaction is difficult to control and the side effectscouldn't be eliminated completely. While the second proposal has anadvantage of that unlike L-asparaginase from Escherichia coli, theL-asparaginases from human may not substantially induce antibodies evenwhen administered to patients, it has a disadvantage of that it is noteasy to obtain the L-asparaginases in a desired amount by the processesdisclosed in Japanese Patent Kokai Nos.320,684/92 and 19,018/80.

Recently, recombinant DNA technology has advanced remarkably. If a DNAwhich encodes a desired polypeptide is once isolated, it is relativelyeasy to obtain a transformant which produces the polypeptide byconstructing a recombinant DNA, comprising the DNA and a self-replicablevector, followed by introducing the recombinant DNA into a host, such asa microorganism, animal- or plant-cell. The polypeptide is obtainable ina desired amount from the culture of the transformant. However, no DNAwhich encodes mammalian L-asparaginase was isolated, and no mammalianL-asparaginase was produced by recombinant DNA techniques.

Therefore, it has been in great demand to isolate DNAs which encodeactive L-asparaginases originating from mammal and establish processesto prepare the L-asparaginases on a large-scale by applying therecombinant DNA techniques to the isolated DNAS.

SUMMARY OF THE INVENTION

In view of foregoing, the first object of the present invention is toprovide a polypeptide which originates from mamma, having L-asparaginaseactivity.

The second object of the present invention is to provide a DNA whichencodes the polypeptide.

The third object of the present invention is to provide a recombinantDNA which containing a DNA which encodes the polypeptide and aself-replicable vector.

The fourth object of the present invention is to provide a transformantobtainable by introducing a DNA which encodes the polypeptide into ahost.

The fifth object of the present invention is to provide a process toprepare the polypeptide by using the transformant.

The sixth object of the present invention is to provide an agent forsusceptive diseases, containing the polypeptide as an effectiveingredient.

The first object of the present invention is attained by polypeptideswhich originate from mammal, having L-asparaginase activity.

The second object of the present invention is attained by DNAs whichencode the polypeptides.

The third object of the present invention is attained by recombinantDNAs containing DNA which encode the polypeptides and a self-replicablevector.

The fourth object of the present invention is attained by transformantsobtainable by introducing the DNAs into appropriate hosts.

The fifth object of the present invention is attained by a process toprepare the polypeptides which comprises culturing the transformants andcollecting the produced polypeptides from the resultant cultures.

The sixth object of the present invention is attained by agents forsusceptive diseases, containing the polypeptides as effectiveingredients.

BRIEF EXPLANATION OF THE ACCOMPANYING DRAWING

FIG. 1 is a scheme of the over lap extension method.

FIG. 2 is a restriction map of the present recombinant DNA pKGPA/WT.

FIG. 3 is a scheme of the preparation of the present recombinant DNApBIgGPA/WT.

FIG. 4 is a restriction map of the present recombinant DNA pBIgGPA/WT.

FIG. 5 is a restriction map of the present recombinant DNApKGPA/D364stp.

FIG. 6 is a restriction map of the present recombinant DNA pKHA/MUT1.

FIG. 7 is a restriction map of the present recombinant DNA pKHA/MUT2.

FIG. 8 is a restriction map of the present recombinant DNA pKHA/MUT3.

FIG. 9 is a restriction map of the present recombinant DNA pKHA/MUT5.

FIG. 10 is a restriction map of the present recombinant DNApBIgGPA/D364stp.

FIG. 11 is a restriction map of the present recombinant DNA pBIgHA/MUT1.

FIG. 12 is a restriction map of the present recombinant DNA pBIgHA/MUT2.

FIG. 13 is a restriction map of the present recombinant DNA pBIgHA/MUT3.

FIG. 14 is a restriction map of the present recombinant DNA pBIgHA/MUT4.

Explanation of the symbols are as follows:

The symbols, “Eco RI”, “Hin dIII”, “Not I” and “Xho I”, indicatecleavage sites by restriction enzymes, Eco RI, Hin dIII, Not I and XhoI, respectively.

The symbols, “D364stp”, “HA/MUT1”, “HA/MUT2”, “HA/MUT3” and “HA/MUT5”,indicate DNAs encoding the present polypeptides.

The symbol “Ptac” indicates a Tac promotor.

The symbol “rrnBT1T2” indicates a region for transcriptionaltermination, derived from a ribosomal RNA operon.

The symbol “AmpR” indicates an ampicillin resistant gene.

The symbol “pBR322ori” indicates a replication origin in Escherichiacoli.

The symbol “Ig sec” indicates a DNA encoding a polypeptide with a signalsequence for secretion of immunoglobulin.

The symbol “Emsv” indicates an enhancer from long terminal repeats ofMoloney Mouse Sarcoma Virus.

The symbol “Pmti” indicates a promotor for Mouse metallothionein I gene.

The symbol “Poly (A)” indicates a polyadenylation signal derived fromSV40 virus.

The symbol “BPVI ” indicates a genome of a bovine paplillomavirus.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors isolated mammalian DNAs encoding L-asparaginasesfirstly in the world, from guinea pig and human, and succeeded inelucidating their nucleotide sequences. The nucleotide sequences of theDNAs from a guinea pig and human are in SEQ ID NOs:15 and 16,respectively. This information is disclosed in Japanese PatentApplication No.42,564/95 (Japanese Patent Kokai No.214,885/96) by thesame applicant of this application. The present invention has been madebased on the above information, and provides the polypeptides whichoriginate from mammal, having L-asparaginase activity.

The polypeptides of the present invention are not restricted to theirsources or origins so far as they originate from mammal and have anL-asparaginase activity. The polypeptides are usually obtainable by theexpression of genes originating from mammal, and usually contain aminoacid sequences of SEQ ID NOs:1 to 3, wherein the symbol “Xaa” in SEQ IDNO:3 means “glutamine” or “arginine”. For example, the polypeptides haveany one of amino acid sequences of SEQ ID NOs:4 to 9. In view of thetechnical level in this field, one or more amino acid residues in SEQ IDNOs:4 to 9 can be substituted relatively easily by different oneswithout substantial defects of the activity. Despite derived from thesame DNA, a variety of polypeptides with an L-asparaginase activity maybe obtained as a result of modifications by endogenous enzymes of thehosts after the DNA expression or modifications during purification ofthe polypeptides, depending on the types of vectors and hosts used toobtain transformants or culturing conditions of the transformants, suchas ingredients, compositions, temperatures or pHs. The wording “avariety of polypeptides” includes the polypeptides with deletions and/oradditions of one or more amino acids at the N-and/or C-termini thereof,or with glycosylations. In view of these, the present polypeptidesinclude not only the polypeptide with any amino acid sequence of SEQ IDNOs:4 to 9 but also their homologues so long as they have anL-asparaginase activity. The present polypeptides express the activitywhen exist in multiple forms, preferably, tetramers.

The polypeptides of the present invention can be usually prepared by therecombinant DNA techniques. In general, the polypeptides are obtainableby culturing transformants containing DNAs encoding the polypeptides andcollecting the produced polypeptides from the resultant cultures. Thetransformants are obtainable by introducing such recombinant DNAs ascontain any one of the nucleotide sequences of SEQ ID Nos:10 to 15 and aself-replicable vector into appropriate hosts. One or more nucleotidesin SEQ ID NOs:10 to 15 can be substituted by different nucleotideswithout substantial changes of the encoding amino acid sequences withrespect to degeneracy of genetic code. To facilitate the expression ofthe DNA in the hosts, one or more nucleotides in nucleotide sequenceswhich encode the polypeptides or their homologues can be appropriatelysubstituted by different ones. Furthermore, nucleotide sequences whichencode and/or don't encode one or more amino acids can be added to the5′- and/or 3′-termini of the nucleotide sequences.

The DNAs encoding the polypeptides of this invention include those fromnatural sources and those by synthesized artificially so far as thepolypeptides expressed by them have an L-asparaginase activity. The DNAscan be wild-type ones, containing the same nucleotide sequences as thosefrom natural sources, and can be their homologues.

Examples of the wild-type DNAs include DNAs containing the nucleotidesequences of SEQ ID NOs:15. The wild-type DNA is obtainable from naturalsources such as guinea pig livers, as disclosed in Japanese PatentApplication No.42,564/95 (Japanese Patent Kokai No.214,885/96) by thesame applicant of this invention: (a) constructing a cDNA library byapplying usual methods to purified poly (A)⁺ RNAs from a guinea pig orhuman liver as materials, (b) applying the plaque hybridization methodto the cDNA library using oligonucleotides as probes synthesizedchemically based on partial amino acid sequences of L-asparaginasepurified from a guinea pig serum, (c) collecting phage clones containingthe DNAs encoding the polypeptides of this invention, and (d)manipulating the collected phage clones in a conventional manner. Thewild-type DNA can be synthesized chemically based on SEQ ID NO:15.

Examples of DNA homologues to the wild-type ones include DNAs containingany nucleotide sequence of SEQ ID NOs:10 to 14. DNA homologuescontaining the nucleotide sequence of SEQ ID NO:10 are obtainable byapplying conventional methods in this field, such as PCR method andmethods for site-directed mutagenesis, to the wild-type DNA of SEQ IDNO: 15 concerning the desired sequence. DNA homologues containing anynucleotide sequence of SEQ ID NOs:11 to 14 are obtainable by the methodssuch as follows: Firstly, A wild-type DNA with the nucleotide sequenceof SEQ ID NO:16 is obtained by the methods as disclosed in JapanesePatent Application No.42,564/95 (Japanese Patent Kokai No.214,885/96) bythe same applicant of this invention, i.e., screening a human liver cDNAlibrary. Subsequently, the wild-type DNA is subjected to conventionalmethods as mentioned above concerning desired sequences to obtain theDNA homologues.

The DNA homologues can be synthesized chemically based on the nucleotidesequences of SEQ ID NOs:10 to 14.

The present DNAs can be generally introduced into hosts as in forms ofrecombinant DNAs. In general, each recombinant DNA comprises one of thepresent DNAs and a self-replicable vector. The recombinant DNAs can beeasily prepared by general recombinant DNA techniques when the DNAs areavailable. Examples of such self-replicable vectors include pKK223-3,pGEX-2T, pRL-λ, pBTrp2 DNA, pUB110, YEp13, Ti plasmid, Ri plasmid,pBI121, pCDM8, pBPV and BCMGSneo. Among these vectors, pKK223-3,pGEX-2T, pRL-λ, pBTrp2 DNA pUB110 are suitably used to express thepresent DNAs in prokaryotic cells such as Escherichia coli and Bacillussp., while YEp13, Ti plasmid, Ri plasmid, pBI121, pCDM8, pBPV andBCMGSneo are suitably used to express the present DNAs in eukaryoticcells such as yeasts and animal- and plant-cells.

To insert the present DNAs into the vectors, conventional methods inthis field can be arbitrarily used. Examples of such methods contain thesteps of (a) cleaving self-replicable vectors with restriction enzymes,(b) introducing the same cleavage sites, by the same restriction enzymesas used to cleave the vectors, to the 5′- and 3′-termini of the presentDNAs by applying polymerase chain reaction to form double-stranded DNAs,(c) cleaving the double-stranded DNAs by the restriction enzymes, and(d) ligating the cleaved vectors with cleaved DNAs by the action of DNAligases. The recombinant DNAs thus obtained can be easily introducedinto appropriate hosts, resulting in limitless replication of the DNAsby culturing the transformants.

The recombinant DNAs according to the present invention can beintroduced into appropriate hosts such as Escherichia coli, Bacillussp., actinomycetes, yeasts and plant- and animal-cells. To introduce theDNAs into Escherichia coli, it can be cultured in the presence of therecombinant DNAs and calcium ion. To introduce them into Bacillus sp.,competent cell methods or protoplast methods can be used. To introducethem into animal-cells, DEAE-dextran methods or electroporation methodscan be used. Desired transformants can be cloned by applyinghybridization methods or by selecting L-asparaginase producing cellsfrom the cultures.

The transformants thus obtained produce the present polypeptidesintracellularly or extracellularly when cultured in nutrient culturemedia. Examples of such media are usually liquid nutrient culture mediawhich generally contain carbon sources, nitrogen sources and minerals,and further contain micronutrients such as amino acids and/or vitaminson demand. The carbon sources usable in the present invention includesaccharides such as starch, starch hydrolysates, glucose, fructose andsucrose. The nitrogen sources usable in the present invention includeorganic and inorganic compounds containing nitrogen, such as ammonia andtheir salts, urea, nitrates, peptone, yeast extract, defatted soy bean,corn steep liquor and beef extract. Cultures containing the presentpolypeptides can be obtained by inoculating the transformants into theabove media, culturing them at temperatures of 25-650° C. at pHs of 5-8for about 1-10 days under aerobic conditions by aeration-agitationmethod, etc.

The cultures can be used intact as agents for susceptive diseases.However, the cultures are usually treated with ultrasonication or cellwall lytic enzymes to disrupt cells, and the present polypeptides areseparated by using techniques such as filtration and centrifugation fromthe cell-disruptants and purified. Alternatively, the polypeptides canbe purified from the culture supernatants obtained by removing cellsfrom the cultures by filtration or centrifugation, etc. The presentpolypeptides can be purified by applying techniques generally used inthis field for protein purifications, such as salting out, dialysis,filtration, concentration, gel filtration chromatography, ion-exchangechromatography, affinity chromatography, hydrophobic chromatography,isoelectric focusing and gel electrophoresis, and if necessary, two ormore of them can be applied combination to the supernatants which areseparated from insoluble substances of cell-disruptants, or to theculture supernatants. The resultant purified solutions polypeptides canbe concentrated and/or lyophilized into liquids or solids depending ontheir final uses.

The following experiments explain the present invention in more detail,and the techniques used therein are conventional ones in this field: Forexample, the techniques are disclosed by J. Sambrook et al. in“Molecular Cloning, A Laboratory Manual”, 2nd edition (1989), publishedby Cold Spring Harbor Laboratory Press, New York, U.S.A., and by MasamiMATSUMURA in “Laboratory Manual for Genetic Engineering” (1988),published by Maruzen Co., Ltd., Tokyo, Japan.

EXPERIMENT 1 Expression of Wild-type DNA Experiment 1-1 Expression ofGuinea Pig Wild-type DNA

Experiment 1-1(a)

Preparation of Guinea Pig Wild-type DNA

A guinea pig wild-type DNA encoding L-asparaginase was prepared by themethod disclosed in Japanese Patent Kokai No.214,885/96 by the sameapplicant of this invention. The DNA had the nucleotide sequence of SEQID NO:15. A DNA having a polypeptide-encoding region in SEQ ID NO:15,i.e., a sequence of containing the nucleotides 20-1,714 in SEQ ID NO:15,is called “GPA/WT DNA” hereinafter, and the expression product thereofwith the amino acid sequence of SEQ ID NO:15 is called “guinea pigwild-type L-asparaginase”. SEQ ID NO:17 shows in parallel the nucleotidesequence SEQ ID NO:49 of GPA/WT DNA and the amino acid sequence (SEQ IDNO:49) encoded thereby.

Experiment 1-1(b)

Preparation of Recombinant DNA

Ten μl of 10×PCR buffer, one μl of 25 mM dNTP mix, one ng of the guineapig wild-type DNA, obtained in Experiment 1-1(a), as a template wereplaced in 0.5 ml reaction tube. The mixture was mixed with, as a sense-and anti-sense-primers, an adequate amount of an oligonucleotidechemically synthesized based on the amino acid sequences near the N- andC-termini of SEQ ID NO:15, volumed up with sterilized distilled water togive a total volume of 99.5 μl, and mixed with 0.5 μl of 2.5 units/μl ofAmpliTaq DNA polymerase. The nucleotide sequence of the sense primer was5′-AATCTCGAGCCACCATGGCGCGCGCATCA-3′ (SEQ ID NO:19) nucleotide sequenceobtained by adding a common nucleotide sequence in animal cells, asshown by M. Kozak in “Nucleic Acid Research”, Vol.15, pp. 8,125-8,148(1987), to the upstream of a region which encodes the N-terminal aminoacid sequence of SEQ ID NO:15 and then adding to the further upstream acleavage site by a restriction enzyme, Xho I. The nucleotide sequence ofthe anti-sense primer was 5′-CTGCGGCCGCTTATCAGATGGCAGGCGGCAC-3′ (SEQ IDNO:20) as a complement to a nucleotide sequence obtained by adding twotermination codons to the downstream of a region which encodes theC-terminus of the amino acid sequence of SEQ ID NO:15 and adding acleavage site by a restriction enzyme, Not I, to the further downstream.The resulting mixture was successively incubated at 94° C. for one min,at 55° C. for one min, and at 72° C. for 3 min, and the series ofincubation was repeated 40-times for PCR to amplify DNA. Thus, a DNAcontaining GPA/WT DNA was obtained and then cleaved by restrictionenzymes of Xho I and Not I to obtain an about 1.7 kbp DNA fragment.Twenty-five ng of the DNA fragment was weighed and mixed with 10 ng of aplasmid vector, “pCDM8”, commercialized by Invitrogen Corporation, SanDiego, U.S.A., which had been cleaved by restriction enzymes of Xho Iand Not I. To the DNA mixture thus obtained was added an equal volume ofthe solution I in “LIGATION KIT VERSION 2” commercialized by TakaraShuzo, Tokyo, Japan, and incubated at 16° C. for 2 hours to obtain areplicable recombinant DNA, “pCGPA/WT”.

The recombinant DNA pCGPA/WT was introduced into an Escherichia coliMC1061/P3 strain, commercialized by Invitrogen Corporation, San Diego,U.S.A., by competent cell method. The transformant thus obtained wasinoculated into L broth medium (pH 7.2) containing 20 μg/ml ampicillinand 10 μg/ml tetracycline followed by cultivation at 37° C. for 18 hoursunder shaking conditions. The transformants were collected from theculture by centrifugation and subjected to conventional alkali-SDSmethod to extract the recombinant DNA pCGPA/WT. The analysis of thepCGPA/WT by an automatic sequencer equipped with a fluorophotometerconfirmed that it contained GPA/WT DNA, which termination codons wereligated to the 3′-terminus and was ligated to the downstream of a CMVpromotor from the 5′- to 3′-termini.

The system using COS-1 (ATCC CRL-1650) as a host, which is a cell linederived from a monkey kidney, was used to express the DNA in thefollowing Experiments 1 and 2. Since the system is for a transientexpression, it has a disadvantage that DNAs introduced intotransformants could not be stable over several days, and thetransformants do not produce the desired polypeptides repeatedly.However, it is known that the number of copies of the desired DNA percell temporally increases to 10⁵ when plasmid vectors having areplication origin derived from SV40 virus, such as the above mentionedpCDM8, are introduced into the COS-1 cells. With this point of view, thesystem has a merit that it quite easily analyzes the desiredDNA-expression product.

Experiment 1-1(c)

Recombinant DNA Expression in COS-1 Cell

In accordance with the DEAE-dextran method reported by Frederick M.Ausubel et al. in “Current Protocols in Molecular Biology” (1987),chapters 9.2.1-9.2.3 and 9.2.5-9.2.6, published by John Wiley and SonsInc., New York, U.S.A., the recombinant DNA pCGPA/WT in Experiment1-1(b) was introduced into COS-1 cells for its expression. To each wellof “3046”, a plastic multiwell plate, with 6 wells of 3.5 cm diameter,commercialized by Becton Dickinson Labware, New Jersey, U.S.A., wasadded 2.5 ml of DME medium, containing 10 v/v % bovine fetal serum and1.8×10⁵ COS-1 cells. The cells were cultured at 37° C. in a 5 v/v % CO₂incubator overnight. After removing the culture supernatant by anaspirator and washing the remaining cells with DME medium containing 50mM Tris-HCl buffer (pH 7.4), each well was charged with 2.5 ml of DMEmedium containing 2.8 μg/ml PCGPA/WT, 50 Mm Tris-HCl (pH 7.4), 0.4 mg/mlDEAE-dextran. and 0.1 mM chloroquine, and incubated at 37° C. for 4hours in a 5 v/v % CO₂ incubator. Thereafter, the culture supernatantwas removed, and the remaining cells in each well were received with 2.5ml of 10 mM phosphate buffered saline (hereinafter abbreviated as “PBS”)containing 10 v/v % DMSO before incubating at ambient temperature for 2minutes. After removing the supernatant and washing the remaining cellswith DME medium containing 50 mM Tris-HCl (pH 7.4), each well wascharged with 2.5 ml of “COS MEDIUM”, commercialized by COSMO BIO CO.LTD., Tokyo, Japan, followed by cultivation at 37° C. for 3 days in a 5v/v % CO₂ incubator to express the desired DNA. As a control, the sameexperiment was carried out using a plasmid vector, pCDM8.

After 3 days' cultivation, the multiwell plates with the cultures weresubjected thrice to a treatment of freezing at −80° C. and thawing atambient temperature to disrupt the cells. The whole cultures weretransferred to centrifugal tubes and centrifuged to remove insolublecomponents after precipitated, followed by obtaining total solublefractions, concentrating the fractions using membranes, and adjustingthe volume of the total soluble fraction per well to give 0.5 ml for thefollowing analyses.

Experiment 1-1(d)

Assay for L-asparaginase Activity

L-Asparaginase activity was expressed by the unit assayed as follows:Samples were placed in 1.5 ml-reaction tubes in 50 μl each and admixedwith 200 μl of 50 mM phosphate buffer (pH 7.0) containing 1.4 mg/mlL-asparagine. After standing at 37° C. for 0, 1, 2, 4, 6 and 16 hours,L-aspartic acid in the reaction mixtures was quantified by an amino acidanalyzer. In parallel, 1.0, 0.5 and 0.25 unit/ml dilutions of anL-asparaginase from Escherichia coli were provided and quantified forL-aspartic acid after incubating at 37° C. for 0 and one hour, and basedon the increased amount of L-aspartic acid, a calibration curve wasdrawn. By plotting on the calibration curve the increased amounts ofL-aspartic acid of the samples, the samples' L-asparaginase activitieswere estimated. The activity of samples with a lower activity wasestimated based on that assayed after 2 hours or more incubation. Oneunit activity of L-asparaginase was defined as the amount that releasesone μmol of ammonia from L-asparagine per minute under the aboveconditions.

The total soluble fractions obtained in Experiment 1-1(c) were treatedsimilarly as above, and expressed their activities as totalL-asparaginase activities that were detected in the soluble fractionsfrom 1.8×10⁵ COS-1 cells. As a result, the activity of the total solublefraction in Experiment 1-1(c) was 0.083 unit, and the control gave noactivity.

Experiment 1-1(e)

Western Blotting

An anti-L-asparaginase antibody was prepared as follows: An oligopeptideof a sequenceGly-Ser-Gly-Asn-Gly-Pro-Thr-Lys-Pro-Asp-Leu-Leu-Gln-Glu-Leu-Arg-Cys (SEQID NO:21) was synthesized chemically in a usual manner. Keyhole LimpedHemocyanin was linked to the C-terminus of the oligopeptide. Theresultant was purified and used to immunize rabbits in a usual manner.The rabbits were immunized 6 times 2 weeks about, then the whole bloodwas collected and subjected to salting out with 50 w/v % ammoniumsulfate to obtain an anti-L-asparaginase anti-serum.

In accordance with the method reported by U. K. Laemli et al. in“Nature”, Vol.227, pp.680-685 (1970), 0.2 ml of the total solublefraction in Experiment 1-1(c) was subjected to 12.5 w/v %SDS-polyacrylamide gel electrophoresis (hereinafter abbreviated as“SDS-PAGE”). The polypeptides migrated were transferred to anitrocellulose membrane and subjected to Western blotting using theabove anti-L-asparaginase anti-serum, in accordance with the methodreported by H. Towbin in “Proceedings of the National Academy ofSciences of the U.S.A.”, Vol.76, pp.4,350-4,354 (1979). For colordevelopment, alkaline phosphatase system was used. Comparing with thecontrol and molecular weight markers, both the identification of bandsspecifically stained in the sample and the measurement of the molecularweight of each subunit of the L-asparaginase were carried out. Themolecular weight markers used were bovine serum albumin (67 kDa),ovalbumin (45 kDa), soy bean trypsin inhibitor (20.1 kDa) andα-lactalbumin (14.4 kDa), and stained with amide black. The totalsoluble fraction in Experiment 1-1(c) gave no clear band.

Experiment 1-1(f)

Measurement of Molecular Weight on Gel Filtration

Two ml of the total soluble fraction in Experiment 1-1(c) was subjectedto gel filtration column chromatography using “HILOAD SUPERDEX 200COLUMN”, with an inner diameter of 16 mm and a length of 60 cm,commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden,equilibrated with PBS. Based on the L-asparaginase activity of theeluted fractions, the molecular weight of the guinea pig wild-typeL-asparaginase in a native form was examined. The molecular weightmarkers used were thyroglobulin (699 kDa), ferritin (440 kDa), catalase(232 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa) andovalbumin (43 kDa). The peak of L-asparaginase activity in the elutedfractions was observed in a position corresponding to a molecular weightof about 300 kDa.

Since no clear band was detected by Western blotting, the molecularweight of the wild-type L-asparaginase in a dissociated form could notbe detected, while the molecular weight in a native form was estimatedto be about 300 kDa based on the result of gel filtration. The molecularweights of L-asparaginase in a native and dissociated form, purifiedfrom guinea pig L-asparaginase in serum, were respectively estimated tobe about 190 kDa on gel filtration and about 43 kDa on SDS PAGE. Asdisclosed in Japanese Patent Kokai No.214,885/96 by the same applicantof the present invention, 3 partial amino acid sequences of a guinea pigL-asparaginase in serum were observed in a region of amino acids 10-236in the sequence of guinea pig wild-type L-asparaginase. While, twoconsensus amino acid sequences essential for the expression ofL-asparaginase activity, i.e., SEQ ID NOs:1 and 2, as proposed by E.Harms in “FEBS letters”, Vol.285, pp.55-58 (1991) based on the resultsof experiments on L-asparaginase derived from Escherichia coli,correspond to the sequences of amino acids 16-19 and 114-118 in theamino acid sequence of the guinea pig wild-type L-asparaginase. In viewof these and the results in Experiment 1-1, the present inventorsestimated that the guinea pig wild-type L-asparaginase may require aregion of amino acids about 1-400 in the amino acid sequence to expressthe activity. In Experiment 2-1, to examine the L-asparaginaseactivities of C-terminal defective mutants as homologues of the guineapig wild-type L-asparaginase, the expression products of DNA homologuesfrom a guinea pig were tested for properties and features.

Experiment 1-2

Expression of Human Wild-type DNA

A human wild-type DNA encoding L-asparaginase was prepared according tothe method in Japanese Patent Kokai No.214,855/96 by the same applicantof the present invention. The DNA had the nucleotide sequence of SEQ IDNO:16. Hereinafter, a DNA having a polypeptide-encoding region in SEQ IDNO:16, i.e., a sequence of nucleotides 93-1,811 in SEQ ID NO:16, wasnamed “HA/WT DNA”, and a polypeptide, as the expression product of HA/WTDNA, having the amino acid sequence of SEQ ID NO:16, may be called“human wild-type L-asparaginase”. SEQ ID NO:18 shows the nucleotidesequence of GPA/WT DNA and the amino acid sequence (SEQ ID NO:50)encoded thereby.

Except for the template and the sense- and anti-sense-primers, PCR wasperformed under the same conditions as used in Experiment 1-1(b). As atemplate, the human wild-type DNA in Experiment 1-2 was used. As asense- and anti-sense-primers, oligonucleotides with sequences of5′-AATCTCGAGCCACCATGGCGCGCGCG GTG-3′ (SEQ ID NO:22) and5′-CTGCGGCCGCTTATCAGACACCAGGCAGCAC-3′ (SEQ ID NO:23) were respectivelyused. The DNA thus amplified was continuously treated with the samemethod as used in Experiment 1-1(b) to prepare a recombinant DNA,“pCHA/WT”. After sequencing, the pCHA/WT was introduced into COS-1 cellsand expressed followed by analyzing the expression product similarly asin Experiment

In contrast to the guinea pig wild-type L-asparaginase, the experimentsystem could not detect the human wild-type L-asparaginase activity. Itwas presumably due to that the human wild-type L-asparaginase had alower specific activity than that of the guinea pig wild-type one, andthis forced to examine the properties of expression products by DNAhomologues from human in Experiment 2-2.

EXPERIMENT 2 Expression of DNA Homoloque Experiment 2-1 Expression ofDNA Homoloque Originating from Guinea Pig

A termination codon was replaced for the nucleotide sequence in aspecific position of the guinea pig wild-type DNA to obtain a DNAhomologue: A DNA was obtained by PCR method by replacing a terminationcodon for a codon of the nucleotides 1,090-1,092 or 1,012-1,014 in SEQID NO:17. Except for the nucleotide sequence of anti-sense primer, PCRwas performed under the same conditions as used in Experiment 1-1(b). Asan anti-sense primer, an oligonucleotide with a sequence of5′-CTGCGGCCGCTTATCATGCCGTGGGCAGTGT-3′ (SEQ ID NO:24) 5′-CTGCGGCCGCTTATCAGCCCAACACGTAGGA-3′ (SEQ ID NO:25) was used to preparethe two-types of DNAs. The amplified DNAs were treated similarly as inExperiment 1-1(b) to obtain recombinant DNAs, “pCGPA/D364stp” and“pCGPA/L338stp”. By sequencing similarly, it was confirmed thatpCGPA/D364stp and pCGPA/L338stp had DNAs, encoding the sequences ofamino acids 1-363 and 1-337 in the guinea pig wild-type L-asparaginase,respectively, and had a termination codon at their 3′-termini free ofintervening sequences. Hereinafter, the polypeptide-encoding regions ofthe DNAs are respectively named “GPA/D364stp DNA” and “GPA/L338stp DNA”.GPA/D364stp DNA and GPA/L338stp DNA were ligated in the downstream of aCMV promoter in the direction from the 5′- to 3′-termini. The DNAsexpression products may be named “guinea pig L-asparaginase homologues”.

The above recombinant DNAs were introduced into COS-1 cells and examinedsimilarly as in Experiment 1-1. As controls, pCGPA/WT and pCDMB inExperiment 1-1(b) were similarly treated and examined. Table 1 shows theresults.

TABLE 1 L-asparaginase Recombinant activity Molecular weight Molecularweight DNA (unit) (kDa) *1 (kDa) *2 PCGPA/WT 0.083 — about 300pCGPA/D364stp 0.228 about 40 about 140 PCGPA/L338stp N.D. *3 about 40 —pCDM8 N.D. *3 — — Note: The symbols “*1”, “*2” and “*3” mean that thevalue was determined by Western blotting, the value was determined bygel filtration, and the activity was not detected, respectively.

As shown in Table 1, the activities of the expression products of GPA/WTDNA and GPA/D364stp DNA were detected, but not for GPA/L338stp DNA.These results suggest that a region of amino acids 1-363 in the guineapig wild-type L-asparaginase may be enough for sufficiently expressingthe L-asparaginase activity. This amino acid sequence, amino acids 1-363in the guinia pig wild-type, is SEQ ID NO:4, and a nucleotide sequencewhich encodes the amino acid sequence is SEQ ID NO:10. The amino acidsequence of the guinea pig wild-type L-asparaginase is SEQ ID NO:5.

EXPERIMENT 2-2 Expression of DNA Homoloque Originating From Human

DNA homologues were prepared by replacing specific codons in the humanwild-type DNA with termination codons or codons for different aminoacids: The DNA homologues were prepared by replacing termination codonsfor the nucleotides 1096-1098 in SEQ ID NO:18 by applying PCR method.Except for the template and the sense- and anti-sense-primers, PCR wasperformed under the same conditions as used in Experiment 1-1(b). As atemplate, the human wild-type DNA in Experiment 1-2 was used. As asense- and anti-sense-primers, the oligonucleotides with sequences of5′-AATCTCGAGCCACCATGGCGCGCGCGGTG-3′ (SEQ ID NO:22) and5′-CTGCGGCCGCTCATTACACCGAGGGTGGCGT-3′ (SEQ ID NO:26) were respectivelyused. The amplified DNA was treated similarly as in Experiment 1-1 toobtain a recombinant DNA, “pCHA/E366stp”, and sequenced. It wasconfirmed that pCHA/E366stp contained a DNA encoding amino acids 1-365in SEQ ID NO:16 and a termination codon at the 3′-terminus free ofintervening sequences. The polypeptide-encoding region was named“HA/E366stp DNA”, hereinafter. HA/E366stp DNA was ligated to thedownstream of a CMV promotor in the direction from the 5′- to3′-termini.

To change specific codons in DNAs into ones for different amino acids,the over lap extension method reported by Robert M. Horton et al. in“Methods in Enzymology”, Vol.217, pp.270-279 (1993), published byAcademic Press, Inc., San Diego, U.S.A., was used. The method issummarized in FIG. 1 and explained as follows: First, mutagenic primersA and B, where the nucleotides to be mutagenized were substituted bydesired different ones complementary to one another, were prepared. Themutagenic primer A was a sense strand, and the mutagenic primer B was ananti-sense strand. A set of 5′- and 3′-terminal primers, which amplifythe whole region of the desired DNA, were prepared, and they wererespectively a sense- and anti-sense-strands. Second, conventional PCRwas performed using the 5′-terminal primer, the mutagenic primer A, andas a template, a DNA with the original nucleotide sequence. In parallel,another PCR as was performed using the same DNA as a template, the3′-terminal primer, and the mutagenic primer B. These two PCRs werenamed “first step PCRs”. Third, two DNAs amplified in the first stepPCRs were mixed with the 5′- and 3′-terminal primers as used in thefirst step PCRs followed by performing PCR as a second step PCR. The twoDNA fragments amplified in the first step PCRs were used as primers andtemplates to generate mutagenized DNAs, while the 5′- and 3′-terminalprimers were used as primers to amplify the mutagenized DNAs. By thismethod, DNAs into which were introduced 7 types nucleotide substituents,i.e., 7 DNA homologues were prepared. The 7 types nucleotidesubstituents and consequent changes of the encoded amino acid sequencesare summarized in Table 2. The template DNA and mutagenic primers A andB used to prepare the 7 DNA homologues were summarized in Table 3. The5′- and 3′-terminal primers were respectively equal to the sense- andanti-sense-primers as used to prepare pCHA/E366stp in Experiment 2-2.

TABLE 2 Nucleotide substitution (upper line) and DNA homologueRecombinant DNA consequential change of amino acid (lower line)* HA/MUT1DNA pCHA/MUT1 C894G, A902G, G952A, G953A and G1096T H298Q, Q301R, G318Nand E366stp HA/MUT2 DNA pCHA/MUT2 C894G, A902G and G1096T H298Q, Q301Rand E366stp HA/MUT3 DNA pCHA/MUT3 C894G, G952A, G953A and G1096T H298Q,G318N and E366stp HA/MUT4 DNA pCHA/MUT4 A902G, G952A, G953A and G1096TQ301R, G318N and E366stp HA/MUT5 DNA pCHA/MUT5 C894G and G1096T H298Qand E366stp HA/MUT6 DNA pCHA/MUT6 A902G and G1096T Q301R and E366stpHA/MUT7 DNA pCHA/MUT7 G952A, G953A and G1096T G318N and E366stp *Numbersin the upper lines in each column mean a nucleotide number in SEQ ID NO:18. Numbers in the lower lines in each column means an amino acidresidue number in SEQ ID NO: 18. Alphabets on the left and right of thenumbers in the upper lines show nucleotides before and after thenucleotide substitution, respectively. Alphabets on the left and rightof the numbers in the lower lines show amino acids before and after thenucleotide substitution, # respectively. The symbol “stp” means that atermination condon was substituted for a codon in the wild-type DNA.Names for the 7 DNA homologues and the recombinant DNAs containing theDNA homologues are shown in parallel.

TABLE 3 Nucleotide sequences of mutagenic primers A (upper DNA homologueTemplate DNA line) and B (lower line)* HA/MUT1 DNA pCHA/MUT7 the same asused for HA/MUT2 DNA preparation the same as used for HA/MUT2 DNApreparation HA/MUT2 DNA pCHA/E366stp 5′-CCCCcGGAGGCAcTGGGT-3′ (SEQ IDNO: 27) 5′-ACCCAgTGCCTCCgGGGG-3′ (SEQ ID NO: 28) HA/MUT3 DNA pCHA/MUT7the same as used for HA/MUT5 DNA preparation the same as used forHA/MUT5 DNA preparation HA/MUT4 DNA pCHA/MUT7 the same as used forHA/MUT6 DNA preparation the same as used for HA/MUT6 DNA preparationHA/MUT5 DNA pCHA/E366stp 5′-CCCCTGGAGGCAcTGGGT-3′ (SEQ ID NO: 29)5′-ACCCAgTGCCTCCAGGGG-3′ (SEQ ID NO: 30) HA/MUT6 DNA pCHA/E366stp5′-CCCCcGGAGGCAGTGGGT-3′ (SEQ ID NO: 31) 5′-ACCCACTGCCTCCgGGGG-3′(SEQ IDNO: 32) HA/MUT7 DNA pCHA/E366stp 5′-GACGttGGCTCCCGCCAT-3′ (SEQ ID NO:33) 5′-ATGGCGGGAGCCaaCGTC-3′ (SEQ ID NO: 34) Note: Small letters meannucleotides which were substituted for those in human wild-type DNA.

The obtained DNA homologues from human were treated similarly as inExperiment 1-1 to obtain recombinant DNAs “pCHA/MUT1”, “pCHA/MUT2”,“pCHA/MUT3”, “pCHA/MUT4”, “pCHA/MUT5”, “pCHA/MUT6” and “pCHA/MUT7”. Theexpression products of the DNA homologues, obtained in Experiment 2-2,may be named “human L-asparaginase homologues”, hereinafter. Aftersequencing, these DNA homologues were introduced into COS-1 cells,followed by expression and assay. As controls, pCHA/WT obtained inExperiment 1-2 and pCDM8 were treated and examined. Signal intensitiesof bands, detected by Western blotting, were evaluated by densitometryto compare quantitatively the expressed products. The results were inTable 4.

TABLE 4 L-asparaginase activity Molecular weight Quantity Molecularweight Recombinant DNA (unit) *1 (kDa) *2 *3 (kDa) *4 pCHA/WT N.D. — — —pCHA/E366stp N.D. about 40 2.3 — pCHA/MUT1 0.021 about 40 0.4 about 140pCHA/MUT2 0.031 about 40 0.9 about 140 pCHA/MUT3 0.009 about 40 0.1about 140 pCHA/MUT4 N.D. about 40 0.2 — pCHA/MUT5 0.006 about 40 1.2about 140 pCHA/MUT6 N.D. about 40 1.9 — pCHA/MUT7 N.D. about 40 0.2 —pCDM8 N.D. — — — Note: The symbols “*1”, “*2”, “*3” and “*4” mean theactivity was not detected, the value was determined by Western blotting,the value indicates the signal intensity of the band detected on Westernblotting and quantified by densitometry, and the value was determined bygel filtration, respectively.

The results in Table 4 indicate that human L-asparaginases both in thewild-type and in the C-terminal defected mutant, i.e., the expressionproduct of HA/E366stp DNA, as the one of the homologues, had a lowerspecific activity than that from guinea pigs. In addition, these resultsindicate that the specific activity of L-asparaginases among those ofpoint mutants, which some of the amino acids inherent to the humanwild-type L-asparaginase were substituted by different ones, increasedto a detectable level. The human DNA homologues such as HA/MUT1,HA/MUT2, HA/MUT3 and HA/MUT5, which the expression products gave adetectable level of activity, have SEQ ID NOs:11 to 14, respectively,and encoding SEQ ID NOs:6 to 9, respectively.

Based on the results in the above experiments, the present inventorsfound that polypeptides from mammal may require the amino acid sequenceof SEQ ID NO:3 (where the symbol “Xaa” meant “glutamine” or “arginine”)to express a detectable level of L-asparaginase activity in theexpression and assay systems in Experiments 1 and 2, in addition toconventionally known as such amino acid sequences of SEQ ID NOs:1 and 2.The animo acid sequence of the guinea pig wild-type L-asparaginasecontains the SEQ ID NO:3 in the region the amino acids 298-302. Examplesof such polypeptides, having all the amino acid sequences of SEQ IDNOs:1 to 3, include those having SEQ ID NOs:4 and 5 from guinea pigs andthose having SEQ ID NOs:6 to 9 from human.

Based on the above findings, the present inventors invented thepolypeptides having L-asparaginase activity. The following examplesexplain the present invention, and the techniques used therein areconventional ones used in the art, and of course, they are notrestrictive to the present invention:

EXAMPLE A-1 Polypeptides Having L-asparaginase Activity

Example A-1(a)

Preparation of Transformant

Ten μl of 10×PCR buffer, one μl of 25 mM dNTP mix, one ng of therecombinant DNA pCGPA/WT DNA obtained in Experiment 1-1 as a template,and an adequate amount of oligonucleotides as a sense- andanti-sense-primers synthesized chemically based on the 5′- and3-terminal sequences of GPA/WT DNA were placed in 0.5 ml reaction tube.The mixture was mixed with sterilized distilled water to give a totalvolume of 99.5 μl, and 0.5 μl of 2.5 units/μl AmpliTaq DNA polymerasewere further added. The sequence of the sense primer was5′-GCGAATTCATGGCGCGCGCATCA-3′ (SEQ ID NO:35) which was a nucleotidesequence obtained by adding a cleavage site by a restriction enzyme, EcoRI, to the upstream of the 5′-terminus of GPA/WT DNA. The sequence ofthe anti-sense primer was 5′-GCAAGCTTTCAGATGGCAGGCGGCAC-3′, (SEQ IDNO:36) which was complementary to a nucleotide sequence prepared byadding a termination codon to the 3′-terminus of GPA/WT DNA and thenadding a cleavage site by a restriction enzyme, Hin dIII, to thedownstream. The above mixture was subjected to 40 cycles of successiveincubations at 94° C. for one min, at 55° C. for one min, and 72° C. for3 min to perform PCR. By cleaving the amplified DNA by restrictionenzymes Eco RI and Hin dIII, a Eco RI-Hin dIII fragment with a length ofabout 1.7 kbp was obtained. Twenty-five ng of the DNA was mixed with 10ng of plasmid vector “pKK2233-3”, commercialized by Pharmacia LKBBiotechnology AB, Uppsala, Sweden, which had been cleaved by restrictionenzymes Eco RI and Hin dill, and then mixed with the solution I in“LIGATION KIT VERSION 2” commercialized by Takara Shuzo Inc., Tokyo,Japan, in an equal volume of the DNA mixture, followed by incubation at16° C. for 2 hours to obtain a replicable recombinant DNA, “pKGPA/WT”.

The recombinant DNA pKGPA/WT was introduced into an Escherichia colistrain “JM105” by the competent cell method. The resulting transformant“J-GPA/WT” was inoculated to L broth medium (pH 7.2) containing 50 μg/mlampicillin and cultured at 37° C. for 18 hours under shaking conditions.The transformants collected by centrifugation from the culture weresubjected to a conventional alkali-SDS method to extract the recombinantDNA pKGPA/WT. As shown in FIG. 2, analysis using an automatic sequencerequipped with a fluorophotometer revealed that GPA/WT DNA of SEQ IDNO:17 ligated to the downstream of a Tac promotor in the direction fromthe 5′- to 3′-termini. In addition, it was confirmed that a terminationcodon was ligated to the downstream of GPA/WT DNA without interveningsequences.

Example A-1(b)

Production of Polypeptide

The transformant J-GPA/WT was inoculated into L broth medium (pH 7.2),containing 50 μg/ml ampicillin, and cultured at 37° C. for 18 hoursunder shaking conditions to obtain a seed culture. Eighteen L of a freshpreparation of the same medium was placed in a 30-L jar fermenter,inoculated with one v/v % of the seed culture, and cultured at 37° C.under aeration-agitation conditions. A portion of the culture was placedin a cuvette with 1-cm in thickness, incubated until the absorbance at awavelength of 650 nm reached to about 1.5, admixed with IPTG to give afinal concentration of 0.1 mM, and incubated for 5 hours. The cellscentrifugally collected from the culture were suspended in a mixturesolution (pH 7.2) containing 139 mM NaCl, 7 mM Na₂HPO₄ and 3 mM NaH₂PO₄,and supersonicated to disrupt the cells, followed by centrifuging theresultant to obtain a supernatant.

Ammonium sulfate was added to the supernatant under ice-chillingconditions to give a concentration of 50 w/v % and then dissolved tohomogeneity. After standing for several minutes, the precipitates werecollected by centrifugation, dissolved in 20 mM Tris-HCl buffer (pH8.0), and dialyzed against a fresh preparation of the same bufferfollowed by applying the dialyzed solution to “Q SEPHAROSE FF COLUMN”,commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden,equilibrated with the same buffer. After washing sufficiently with thesame buffer, the column was fed with a linear gradient buffer of NaClincreasing from 0 M to 0.5 M in 20 mM Tris-HCl buffer (pH 8.0). Thefractions eluted at about 0.1-0.3 M NaCl were collected, and the solventwas replaced with 10 mM sodium-phosphate buffer (pH 7.5) whileconcentrating with membranes. The concentrated solution was then appliedto “L-ASPARAGINE AGAROSE”, commercialized by Sigma Chemical Co., St.Louis, U.S.A., equilibrated with the same buffer. After washing with thesame buffer, 10 mM sodium phosphate buffer (pH 7.5) containing 0.5 MNaCl was fed to the column for elution. The eluted fractions were pooledand concentrated by using a membrane. The concentrate was applied to“HILOAD SUPERDEX 200 COLUMN”, commercialized by Pharmacia LKBBiotechnology AB, Uppsala, Sweden, equilibrated with Tris-HCl buffer (pH8.0) containing 10 v/v % glycerol, and eluted from the column. Theeluted fractions, containing substances with a molecular weight of about300 kDa, were collected to obtain a purified polypeptide with a purityof 90% or more in a yield of about 0.1 mg/ml culture.

Example A-1(c)

Physicochemical Property of Polypeptide

The purified polypeptide in the above was analyzed to determine thephysicochemical properties: The molecular weight of the purifiedpolypeptide in a native form was determined by gel filtration similarlyas in Experiment 1-1(e). The peak for L-asparaginase activity of theeluted fractions was found at a position corresponding to a molecularweight of about 300 kDa. The molecular weight of the purifiedpolypeptide in a dissociated form was determined by SDS-PAGE as used inExperiment 1-1(e). The main band was observed at a positioncorresponding to a molecular weight of about 50±10 kDa. The resultsindicate that the purified polypeptide exists in a multimer as a nativeform. Considering errors in measurement by the above methods and thefact that all the known L-asparaginases from Escherichia coli. etc.,other than mammal, exist in a tetrameric form, it can be estimated thatthe purified polypeptide exists in a tetrameric form. The method as usedin Experiment 1-1(d) confirmed that the purified polypeptide has anL-asparaginase activity.

Example A-2(a)

Preparation of Transformant

FIG.3 summarizes the procedures to prepare transformants. PCR wasperformed under the same conditions as used in Example A-1(a) except forthe nucleotide sequences of a sense- and anti-sense-primers. As thesense- and anti-sense-primers, oligonucleotides with the nucleotidesequences of 5′-A GTGAATTCGGAGGTTCAGATGGCGCGCGCATCA-3′ (SEQ ID NO:32)and 5′-CTGCGGCCGCTCAGATGGCAGGCGGCAC-3′ (SEQ ID NO:38) were respectivelyused. The DNA thus amplified was cleaved by restriction enzymes Eco RIand Not I to obtain an about 1.7 kbp Eco RI-Not I fragment. Seventy ngof the DNA fragment was mixed with 50 ng of a plasmid vector, “pBPV”,commercialized by Pharmacia LKB Biotechnology AB, Uppsala, Sweden,cleaved in advance by restriction enzymes Xho I and Not I, and 25 ng ofeach of 4 oligonucleotides as linkers with nucleotide sequences of5′-TCGAGCCACCATGAAGTGTTCGTGGGTTATT-3′ (SEQ ID NO:39),5-TTCTTCCTGATGGCCGTAGTGACAGGAGTG-3′ (SEQ ID NO:40),5′-AATTCACTCCTGTCACTACGGCCATCAGGA-3′ (SEQ ID NO:41) and5′-AGAAAATAACCCACGAACACTTCATGGTGGC-3′ (SEQ ID NO:42). Theoligonucleotides for linkers were synthesized in a usual manner and usedafter reacted with T4 polynucleotide kinase, commercialized by PharmaciaLKB Biotechnology AB, Uppsala, Sweden, and purified byethanol-precipitation. To the DNA mixture was added the solution I in“LIGATION KIT VERSION 2”, commercialized by Takara Shuzo, Tokyo, Japan.The mixture was incubated at 16° C. for 2 hours to obtain a replicablerecombinant DNA “pBIgGPA/WT”.

The recombinant DNA pBIgGPA/WT was introduced into an Escherichia coliHB101 strain by the competent cell method. The transformant thusobtained was inoculated into L broth medium (pH 7.2) containing 50 μg/mlampicillin followed by cultivation at 37° C. for 18 hours under shakingconditions. The transformants, collected by centrifuging the resultingculture, were subjected to a conventional alkali-SDS method to extractthe recombinant DNA pBIgGPA/WT. The nucleotide sequence analysis usingan automatic sequencer confirmed that the recombinant DNA pBIgGPA/WT hadthe structure in FIG. 4: A DNA encoding a polypeptide containing asignal sequence for immunoglobulin secretion, as shown by D. F. Stern etal. in “Science”, Vol.235, pp.321-324 (1984), i.e., “Ig sec DNA” wasligated to the downstream of a region for transcriptional regulation,comprising an enhancer derived from long terminal repeats of MoloneyMouse Sarcoma Virus (Emsv), and a promotor derived from Mousemetallothionein I gene (Pmti). Furthermore, GPA/WT DNA was ligated inthe same frame to the downstream of the Ig sec DNA in the direction fromthe 5′- to 3′-termini of GPA/WT DNA. It was also confirmed that atermination codon exists in the 3′-terminus of GPA/WT DNA withoutintervening sequences.

The recombinant DNA pBIgGPA/WT was introduced into a cell line C127(ATCC CRL-1616), derived from a mouse, by using a lipofectin® reagentcommercialized by Life Technologies, Inc., Gaitherburg, U.S.A.,according to the attached protocol. The transformants with therecombinant DNA were selected based on the lack ofproliferation-regulatory ability, i.e., focus-forming ability, as afirst selection. The cells around those containing foci were collectedusing sterilized filter papers and subjected to a conventional limitingdilution method to form single cells which were then selected dependingon the productivity of L-asparaginase, as final selection. Thus, atransformant, “C-GPA/WT”, was obtained.

Example A-2(b)

Production of Polypeptide

The transformant C-GPA/WT was inoculated into a well of “3046”, aplastic multiwell plate with 6 wells, 3.5 cm in diameter, commercializedby Becton Dickinson Labware, New Jersey, U.S.A., with DME mediumcontaining 10 v/v % bovine fetal serum, and cultured to be confluent asa seed culture. Some of the cells, scraped by treatment with trypsin,were inoculated as seed cells into each of the multiwell plates whichwere charged with the fresh preparation of the same medium and cultured.After repeating manipulations similarly as in the above and with scaleup to increase the cell number, the cells were subjected to aconventional continuous culture using 50 of 150 cm² culture flasks. Theresulting culture supernatants of a volume of 100 l was collected andtreated with similar methods for treating the supernatant from thecell-disruptants in Example A-1(b): salting out with ammonium sulfate,the chromatography of the solution of the precipitates using Q SEPHAROSEFF COLUMN, the chromatography of the eluted fractions using L-ASPARAGINEAGAROSE, and the chromatography of the eluted fractions using HILOADSUPERDEX 200 COLUMN. Consequently, a purified polypeptide with a purityof 90% or more was obtained in a yield of about one μg/ml-culture.

Example A-2(c)

Physicochemical Property of Polypeptide

By testing similarly as in Example A-1(c), it was confirmed that thepurified polypeptide thus obtained had equivalent physicochemicalproperties with the that obtained in Example A-1(b).

Example A-3(a)

Preparation of Transformant

PCRs were performed under the same conditions in Example A-1(a) exceptfor the template and the sense- and anti-sense-primers. The DNA thusobtained were treated similarly as in Example A-1(a) to preparerecombinant DNAs, “pKGPA/D364stp”, “pKHA/MUT1”, “pKHA/MUT2”, “pKHA/MUT3”and “pKHA/MUT5”. Table 5 summarizes template DNAs and nucleotidesequences of a sense- and anti-sense-primers which were used to preparethe each recombinant DNAS. By sequencing similarly as in Example A-1(a),the structures of these recombinant DNAs were confirmed as shown inFIGS. 5 to 9.

TABLE 5 Nucleotide sequences of sense (upper line) Recombinant DNATemplate DNA and anti-sense (lower line) primers* pKGPA/D364stppCGPA/D364stp 5′-GCGAATTCATGGCGCGCGCATCA-3′ (SEQ ID NO: 35)5′-GCAAGCTTTCATGCCGTGGCCAGTGT-′ (SEQ ID NO: 43) pKHA/MUT1 pCHA/MUT15′-GCGAATTCATGGCGCGCGCGGTG-3′ (SEQ ID NO: 44)5′-GCAAGCTTTCACACCGAGGGTGGCGT-3′ (SEQ ID NO: 45) pKHA/MUT2 pCHA/MUT2 thesame as used for pKHA/MUT1 preparation the same as used for pKHA/MUT1preparation pKHA/MUT3 pCHA/MUT3 the same as used for pKHA/MUT1preparation the same as used for pKHA/MUT1 preparation pKHA/MUT5pCHA/MUT5 the same as used for pKHA/MUT1 preparation the same as usedfor pKHA/MUT1 preparation *Italics in the upper line in each column meanthe 5′-terminal nucleotide sequence of a DNA encoding L-asparaginase,and those in the lower line mean the complementary sequence to the3′-terminus of the DNA, wherein the L-asparaginese originates from aguinea pig or human.

The recombinant DNAs were treated according to the methods as in ExampleA-1(a) to obtain transformants, “J-GPA/D364stp”, “J-HA/MUT1”,“J-HA/MUT2”, “J-HA/MUT3” and “J-HA/MUT5”.

Example A-3(b)

Production of Polypeptide

The transformants obtained in Example A-3(a) were treated according tothe methods similarly as in Example A-1(b): cultivation, disrupting theresulting cells, the precipitations of the cell-disruptants withammonium sulfate, the chromatography of the precipitate solutions usingQ SEPHAROSE FF COLUMN, and the chromatography of the eluted fractionsusing L-ASPARAGINE AGAROSE in that order. The eluted fractions thusobtained were concentrated using membranes similarly as in ExampleA-1(b) followed by subjecting the chromatography using HILOAD SUPERDEX200 COLUMN to collect the eluted fractions with a molecular weight ofabout 140 kDa. Each system yielded the purified polypeptide with apurity of 90% or more in a yield of about 0.1 mg/ml-culture. Thesepurified polypeptides were analyzed by the methods as in Example A-1(c)to examine their physicochemical properties. Table 6 shows the resultscombined with those in Example A-1(c).

TABLE 6 Transformant, producing Molecular weight Molecular weightL-asparaginase the polypeptide (kDa) *1 (kDa) *2 activity J-GPA/WT about300 about 50 ± 10 + J-GPA/D364stp about 140 about 40 + J-HA/MUT1 about140 about 40 + J-HA/MUT2 about 140 about 40 + J-HA/MUT3 about 140 about40 + J-HA/MUT5 about 140 about 40 + Note) The symbols “*1” and “*2” meanthat the value was determined by gel filtration, and the value wasdetermined by SDS-PAGE, respectively.

Table 6 indicates that all of the present polypeptides, expressed inEscherichia coli and purified, expressed an L-asparaginase activity.Furthermore, table 6 indicates the that the polypeptides formedtetramers.

Example A-4(a)

Preparation of Transformants

PCRs were performed under the same conditions in Example A-1(a) exceptfor the template and the sense- and anti-sense-primers. DNAs thusobtained were ligated with the same linkers as used in Example A-2(a)under the same conditions as in Example A-2(a) to obtain recombinantDNAs, “pBIgGPA/D364stp”, “pBIgHA/MUT1”, “pBIgHA/MUT2”, “pBIgHA/MUT3” and“pBIgHA/MUT5”. Table 7 summarizes template DNAs and nucleotide sequencesof sense- and anti-sense-primers which were used to prepare the eachrecombinant DNAs. By sequencing similarly as in Example A-1(a), thestructures of these recombinant DNAs were confirmed as shown in FIGS. 10to 14.

TABLE 7 Nucleotide sequences of sense (upper line) Recombinant DNATemplate DNA and anti-sense (lower line) primers* pBIgGPA/D364stppCGPA/D364stp 5′-GTGAATTCGGAGGTTCAGATGGCGCGCGCATCA-3′ (SEQ ID NO: 37)5′-CTGCGGCCGCTCATGCCGTGGGCAGTG-3′ (SEQ ID NO: 46) PBIgHA/MUT1 pCHA/MUT15′-CTGAATTCGGAGGTTCAGATGGCGCGCGCGGTG-3′ (SEQ ID NO: 47)5′-CTGCGGCCGCTCACACCGAGGGTGGCG-3′ (SEQ ID NO: 48) pBIgHA/MUT2 pCHA/MUT2the same as used for pBIgHA/MUT1 preparation the same as used forpBIgHA/MUT1 preparation pBIgHA/MUT3 pCHA/MUT3 the same as used forPBIgHA/MUT1 preparation the same as used for pBIgHA/MUT1 preparationpBIgHA/MUT5 pCHA/MUT5 the same as used for pBIgHA/MUT1 preparation thesame as used for pBIgHA/MUT1 preparation Note) *Italics in the upperline in each column mean the 5′-terminal nucleotide sequence of a DNAencoding L-asparaginase, and those in the lower line mean thecomplementary sequence to the 3′-terminus of the DNA, wherein theL-asparaginese originates from a guinea pig or human.

The recombinant DNAS thus obtained were treated similarly as in ExampleA-2(a) to obtain transformants, “C-GPA/D364stp”, “C-HA/MUT1”,“C-HA/MUT2”, “C-HA/MUT3” and “C-HA/MUT5”.

Example A-4(b)

Production of Polypeptide

The transformants obtained in Example A-4(a) were cultured according tothe methods as in Example A-2(b), and the resulting culture supernatantswere treated with similar methods for treating the supernatants from thecell-disruptants in Example A-1(b): the precipitations of culturesupernatants with ammonium sulfate, the chromatography of theprecipitate solutions using Q SEPHAROSE FF COLUMN, and thechromatography of the eluted fractions using L-ASPARAGINE AGAROSE inthat order. The eluted fractions thus obtained were concentrated usingmembranes similarly as in Example A-1(b) followed by subjecting thechromatography using HILOAD SUPERDEX 200 COLUMN to collect the elutedfractions with a molecular weights of about 140 kDa. Each of thesesystems yielded the purified polypeptide with a purity of 90% or more ina yield of about one μg/ml-culture. These purified polypeptides wereanalyzed by the methods as in Example A-1(c) to examine theirphysicochemical properties. Table 8 shows the results combined withthose in Example A-3.

TABLE 8 The polypeptide- producing Molecular weight Molecular weightL-asparaginase transformant (kDa) *1 (kDa) *2 activity J-GPA/WT about300 about 50 ± 10 + J-GPA/D364stp about 140 about 40 + J-HA/MUT1 about140 about 40 + J-HA/MUT2 about 140 about 40 + J-HA/MUT3 about 140 about40 + J-HA/MUT5 about 140 about 40 Note) The symbols “*1” and “*2” meanthat the value was determined by gel filtration, and the value wasdetermined by SDS-PAGE, respectively.

Table 8 indicates that all of the present polypeptides, expressed inmammalian cells and purified, expressed an L-asparaginase activity.Furthermore, table 8 indicates the polypeptides formed tetramers.

As shown in above Example A, each of the polypeptides according to thepresent invention expresses an L-asparaginase activity. Therefore, thepresent agent for susceptive diseases hydrolyze L-asparagine in patientsto exert therapeutic and preventive effects on L-asparaginase-susceptivediseases when administered to human. The wording “susceptive diseases”as referred in the present specification means diseases in general whichare caused by the existence of tumor cells dependent on L-asparagine:For example, leukemias such as acute leukemia, an acute transformationof chronic leukemia and T-lymphocytic leukemia, and malignant tumorssuch as Hodgkin's diseases and non-Hodgkin's diseases. The present agentfor susceptive diseases possesses thus the uses as anti-tumor agents fortreating and/or preventing such susceptive diseases as above. Althoughit varies dependently on the types of agents used for such purposes andsusceptive diseases to be treated, the present agent is generallyprocessed into an agent in the form of a liquid, a paste or a solidwhich contains the polypeptides in an amount of 0.000001-100 w/w %,preferably, 0.0001-100 w/w A, on a dry solid basis.

The present agent can be used intact or processed into compositions bymixing with one or more selected from the group consisting ofphysiologically-acceptable carriers, excipients, solvents, buffers andstabilizers, and further, if necessary, other biologically-activesubstances and other agents. For example, “Iyakuhin-Tenkabutsu-Jiten(The Dictionary of Pharmaceutical Excipients)” (1994), edited by JapanPharmaceutical Excipients Council, Tokyo, Japan, published byYakujinippo LTD., Tokyo, Japan and “Iyakuhin-Tenkabutsu-Jiten-Tsuiho1995 (Suppliment for The Dictionary of Pharmaceutical Excipients)”(1995), edited by Japan Pharmaceutical Excipients Council, Tokyo, Japan,published by Yakujinippo LTD., Tokyo, Japan, mention the embodiments ofsuch carriers, excipients, solvents, buffers and stabilizers. Examplesof such other biologically-active substances and other agents includeinterferon-α, interferon-β, interferon-γ, interleukin 1, interleukin 2,interleukin 3, TNF-α, TNF-β, GM-CSF, carboquone, cyclophosphamide,aclarbicin, thiotepa, busulfan, ancitabine, cytarabine, fluorouracil,5-fluoro-1-(tetrahydro-2-furyl)uracil, methotrexate, actinomycin D,chromomycin A3, daunorubicin, doxorubicin, bleomycin, mercaptopurine,prednisolone, mitomycin C, vincristine, vinblastine, radio goldcolloidal, Krestin®, picibanil, lentinan and Maruyama vaccine.

The present agent for susceptive diseases includes those in a unit doseform which means a physically separated and formed medicament suitablefor administration, and contains the polypeptides in a daily dose or ina dose from 1/40 to several folds (up to 4 folds) of the daily dose.Examples of such medicaments are injections, liquids, powders, granules,tablets, capsules, sublinguals, ophthalmic solutions, nasal drops andsuppositories.

The present agent can be administered to patients orally orparenterally. In both administrations, the agent exerts a satisfactoryeffect in the treatment and/or the prevention for the susceptivediseases. Although it varies dependently on the types of susceptivediseases and their symptoms, the agent can be orally administered topatients or parenterally administered to patients' intradermal tissues,subcutaneous tissues, muscles, and veins at a dose as amounts of thepolypeptides in the range of about 0.1 μg-500 mg/shot, preferably, about0.1-100 mg/shot, 1-4 times/day or 1-7 times/week, for one day to oneyear. The present agent for susceptive diseases further includes theforms by applying gene therapy. When a transformant into which the DNAsencoding the polypeptides of this invention are introduced areadministered to patients to express in them, they exert equivalenteffects as above administrations. For example, “Jikken-Igaku Bessatsu,Bio-manual Up Series, Idenshi-Chiryo-No-Kisogijutsu (Basic Techniquesfor Gene Therapy)” (1996), edited by Takashi SHIMADA, Izumi SAITO andTakaya OZAWA, published by Yodosha, Tokyo, Japan, details the generalprocedures for the gene therapy.

The biological activities and acute toxicity of the present polypeptidesare explained based on Experiment 3 and 4 below, respectively.

EXPERIMENT 3 Bioloqical Activity

Experiment 3-1

Antitumor Effect in Vitro

A human histocytic lymphoma cell line U937 (ATCC CRL-1593), and a cellline Molt4 (ATCC CRL-1582), derived from human T lymphoblasts, weresubcultured in RPMI 1640 medium containing 10 v/v % bovine fetal serum.The cells collected by centrifugation from each subculturing system inlogarithmic phase were suspended in the same medium to give aconcentration of 2×10⁵ cells/ml. Every one ml of the each cellsuspension was charged into each of 13 wells of multiwell plates with 24wells, “3047”, commercialized by Becton Dickinson Labware, New Jersey,U.S.A. After each of dilutions of 12 types of the purified polypeptidesprepared in Example A-1 to A-4 with PBS was further charged into theeach well, the cells were cultured at 37° C. for 72 hours in a 5 v/v %CO₂ incubator. The final concentration of each of the purifiedpolypeptides was one unit/ml as an L-asparaginase activity. As acontrol, after charged with equivalent volume of PBS, the cells werecultured correspondingly. The cells were collected after the cultivationto stain cells died with trypan blue. Cell survival ratio in eachsystems with the purified polypeptides was compared with that in thecontrol. All of the cell survival ratios with the purified polypeptideswere significantly lower than that in the control. These resultsindicate that all of the present polypeptides, obtained in Examples A-1to A-4, have cytotoxicity to U937 and Molt4.

Experiment 3-2

Antitumor Effect in Vivo

For model mice were used C3H mice wherein a mouse lymphoma cell line6C3HED, registered in Cell Resource Center for Biomedical Research,Institute of Development Aging and Cancer, Tohoku University, Sendai,Japan, was transplanted with passages by subcutaneous injections attheir sides in a range of 1×107 cells/body every 8 days in usual manner.To the model mice were administered the purified polypeptides obtainedin Example A-1 to A-4 in the range of 400 unit/body by venoclyses everyday from fourth to seventh days after transplanted with the cells.Dimensions of the tumors were observed with naked eyes on fourth andeighth day after the transplantations. The purified polypeptides wereadministered after diluted with 0.15 M NaCl and filtrated with membranefilters, 0.45 μm in pore size, commercialized by Millipore Corp.,Bedford, U.S.A. As a control, 0.15 M NaCl was treated correspondingly.While significant enlargements of the tumors were observed in thecontrol, significant involutions or disappearances of the tumors wereobserved in mice administered with the polypeptides. These resultsindicates that all of the present polypeptides, obtained in Examples A-1to A-4, are able to cure the tumors of model mice.

Experiment 4

Acute Toxicity

The purified polypeptides obtained in Examples A-1 to A-4 wereseparately administered to 8-week-old mice percutaneously, perorally orintraperitoneally according to conventional manner. The LD₅₀ of all thepolypeptides was about 100 mg/kg or higher independently of theadministration routes. These results evidenced that the presentpolypeptides could be safely incorporated into pharmaceuticals foradministering human.

The following examples explain the present agent for susceptivediseases.

EXAMPLE B-1 Solution

The purified polypeptides obtained in Examples A-1 to A-4 wereseparately dissolved to give a concentration of 0.1 mg/ml inphysiological saline containing one w/v % human serum albumin as astabilizer, and sterilized with membrane filters according toconventional manner to obtain solutions.

All of the products have satisfactory stabilities and can be used asinjections, ophthalmic solutions, collunarium in the treatment and/orthe prevention of susceptive diseases including a malignant tumor, acuteleukemia, malignant lymphoma, an acute transformation of chronicleukemia, T-lymphocytic leukemia.

EXAMPLE B-2 Solution

The purified polypeptides obtained in Examples A-1 to A-4 wereseparately dissolved to give a concentration of 0.1 mg/ml inphysiological saline containing one w/v % glycerol as a stabilizer, andsterilized with membrane filters according to conventional manner toobtain solutions.

All of the products have satisfactory stabilities and can be used asinjections, ophthalmic solutions, collunarium for the treatment and/orthe prevention of susceptive diseases including a malignant tumor, acuteleukemia, malignant lymphoma, an acute transformation of chronicleukemia and T-lymphocytic leukemia.

EXAMPLE B-3 Dry Injection

The purified polypeptides obtained in Examples A-1 to A-4 wereseparately dissolved to give a concentration of 50 mg/ml inphysiological saline containing one w/v % purified gelatin as astabilizer, and the solutions were sterilized with membrane filtersaccording to conventional manner. One ml aliquots of the sterilizedsolutions were distributed to vials, lyophilized and cap sealed.

All of the products have satisfactory stabilities and can be used as dryinjections for the treatment and/or the prevention of susceptivediseases including a malignant tumor, acute leukemia, malignantlymphoma, an acute transformation of chronic leukemia and T-lymphocyticleukemia.

EXAMPLE B-4 Ointment

“HI-BIS-WAKO 104”, a carboxyvinyl polymer commercialized by Wako PureChemicals, Tokyo, Japan, and a purified trehalose were dissolved insterilized distilled water to give concentrations of 1.4 w/w % and 2.0w/w %, respectively, and the purified polypeptides obtained in ExamplesA-1 to A-4 were separately mixed to homogeneity in the solutionsfollowed by adjusting the pH of the resulting solutions to pH 7.2 toobtain pastes containing about one mg/g of the polypeptides.

All of the products have satisfactory spreadabilities and stabilities,and can be used as ointments for treating and/or preventing susceptivediseases including a malignant tumor, acute leukemia, malignantlymphoma, an acute transformation of chronic leukemia and T-lymphocyticleukemia.

EXAMPLE B-5 Tablet

Any one of the purified polypeptides obtained in Examples A-1 to A-4 andLUMIN, i.e. [bis-4-(1-ethylquinoline)][γ-4′-(1-ethylquinoline]pentamethionine cyanine, as a cell activator were mixed to homogeneitywith “FINETOSED®”, an hydrous crystalline α-maltose commercialized byHayashibara Co., Ltd., Okayama, Japan, and the mixtures were tablettedby tabletting machine to obtain tablets, about 200 mg weight each,containing the polypeptide and the LUMIN, about 5 mg each.

All of the products have satisfactory swallowing abilities, stabilitiesand cell activating activities, and can be used for treating and/orpreventing susceptive diseases including a malignant tumor, acuteleukemia, malignant lymphoma, an acute transformation of chronicleukemia and T-lymphocytic leukemia.

The present invention is based on the findings of polypeptides whichoriginate from mammal, having L-asparaginase activity. The polypeptidesare substances which have revealed amino acid sequences totally, andstable activities to hydrolyze L-asparagine. Therefore, the polypeptidesexert satisfactory effects in the treatment and/or the prevention fordiseases caused by tumor cells dependent on L-asparagine.

The polypeptides originate from mammal, so that they have lowantigenicities to human and don't cause serious side effects even whenadministered in large amounts or continuously. Therefore, thepolypeptides have the advantage that they can exert desired effectswithout restricted controls on patients' sensitivities.

The polypeptides thus valuable can be produced in desired amounts usingthe present DNAs encoding them.

Thus, the present invention is a significant invention which has aremarkable effect and gives a great contribution to this field.

While there has been described what is at present considered to be thepreferred embodiments of the present invention, it will be understoodthe various modifications may be made therein, and it is intended tocover in the appended claims all such modifications as fall within thetrue spirits and scope of the invention.

50 4 amino acids amino acid single linear peptide 1 Thr Gly Gly Thr 1 5amino acids amino acid single linear peptide 2 His Gly Thr Asp Thr 1 5 5amino acids amino acid single linear peptide 3 Gln Cys Leu Xaa Gly 1 5363 amino acids amino acid single linear peptide 4 Met Ala Arg Ala SerGly Ser Glu Arg His Leu Leu Leu Ile Tyr Thr 1 5 10 15 Gly Gly Thr LeuGly Met Gln Ser Lys Gly Gly Val Leu Val Pro Gly 20 25 30 Pro Gly Leu ValThr Leu Leu Arg Thr Leu Pro Met Phe His Asp Lys 35 40 45 Glu Phe Ala GlnAla Gln Gly Leu Pro Asp His Ala Leu Ala Leu Pro 50 55 60 Pro Ala Ser HisGly Pro Arg Val Leu Tyr Thr Val Leu Glu Cys Gln 65 70 75 80 Pro Leu LeuAsp Ser Ser Asp Met Thr Ile Asp Asp Trp Ile Arg Ile 85 90 95 Ala Lys IleIle Glu Arg His Tyr Glu Gln Tyr Gln Gly Phe Val Val 100 105 110 Ile HisGly Thr Asp Thr Met Ala Phe Gly Ala Ser Met Leu Ser Phe 115 120 125 MetLeu Glu Asn Leu His Lys Pro Val Ile Leu Thr Gly Ala Gln Val 130 135 140Pro Ile Arg Val Leu Trp Asn Asp Ala Arg Glu Asn Leu Leu Gly Ala 145 150155 160 Leu Leu Val Ala Gly Gln Tyr Ile Ile Pro Glu Val Cys Leu Phe Met165 170 175 Asn Ser Gln Leu Phe Arg Gly Asn Arg Val Thr Lys Val Asp SerGln 180 185 190 Lys Phe Glu Ala Phe Cys Ser Pro Asn Leu Ser Pro Leu AlaThr Val 195 200 205 Gly Ala Asp Val Thr Ile Ala Trp Asp Leu Val Arg LysVal Asn Trp 210 215 220 Lys Asp Pro Leu Val Val His Ser Asn Met Glu HisAsp Val Ala Leu 225 230 235 240 Leu Arg Leu Tyr Pro Gly Ile Pro Ala SerLeu Val Arg Ala Phe Leu 245 250 255 Gln Pro Pro Leu Lys Gly Val Val LeuGlu Thr Phe Gly Ser Gly Asn 260 265 270 Gly Pro Ser Lys Pro Asp Leu LeuGln Glu Leu Arg Ala Ala Ala Gln 275 280 285 Arg Gly Leu Ile Met Val AsnCys Ser Gln Cys Leu Arg Gly Ser Val 290 295 300 Thr Pro Gly Tyr Ala ThrSer Leu Ala Gly Ala Asn Ile Val Ser Gly 305 310 315 320 Leu Asp Met ThrSer Glu Ala Ala Leu Ala Lys Leu Ser Tyr Val Leu 325 330 335 Gly Leu ProGlu Leu Ser Leu Glu Arg Arg Gln Glu Leu Leu Ala Lys 340 345 350 Asp LeuArg Gly Glu Met Thr Leu Pro Thr Ala 355 360 363 565 amino acids aminoacid linear peptide 5 Met Ala Arg Ala Ser Gly Ser Glu Arg His Leu LeuLeu Ile Tyr Thr 1 5 10 15 Gly Gly Thr Leu Gly Met Gln Ser Lys Gly GlyVal Leu Val Pro Gly 20 25 30 Pro Gly Leu Val Thr Leu Leu Arg Thr Leu ProMet Phe His Asp Lys 35 40 45 Glu Phe Ala Gln Ala Gln Gly Leu Pro Asp HisAla Leu Ala Leu Pro 50 55 60 Pro Ala Ser His Gly Pro Arg Val Leu Tyr ThrVal Leu Glu Cys Gln 65 70 75 80 Pro Leu Leu Asp Ser Ser Asp Met Thr IleAsp Asp Trp Ile Arg Ile 85 90 95 Ala Lys Ile Ile Glu Arg His Tyr Glu GlnTyr Gln Gly Phe Val Val 100 105 110 Ile His Gly Thr Asp Thr Met Ala PheGly Ala Ser Met Leu Ser Phe 115 120 125 Met Leu Glu Asn Leu His Lys ProVal Ile Leu Thr Gly Ala Gln Val 130 135 140 Pro Ile Arg Val Leu Trp AsnAsp Ala Arg Glu Asn Leu Leu Gly Ala 145 150 155 160 Leu Leu Val Ala GlyGln Tyr Ile Ile Pro Glu Val Cys Leu Phe Met 165 170 175 Asn Ser Gln LeuPhe Arg Gly Asn Arg Val Thr Lys Val Asp Ser Gln 180 185 190 Lys Phe GluAla Phe Cys Ser Pro Asn Leu Ser Pro Leu Ala Thr Val 195 200 205 Gly AlaAsp Val Thr Ile Ala Trp Asp Leu Val Arg Lys Val Asn Trp 210 215 220 LysAsp Pro Leu Val Val His Ser Asn Met Glu His Asp Val Ala Leu 225 230 235240 Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ser Leu Val Arg Ala Phe Leu 245250 255 Gln Pro Pro Leu Lys Gly Val Val Leu Glu Thr Phe Gly Ser Gly Asn260 265 270 Gly Pro Ser Lys Pro Asp Leu Leu Gln Glu Leu Arg Ala Ala AlaGln 275 280 285 Arg Gly Leu Ile Met Val Asn Cys Ser Gln Cys Leu Arg GlySer Val 290 295 300 Thr Pro Gly Tyr Ala Thr Ser Leu Ala Gly Ala Asn IleVal Ser Gly 305 310 315 320 Leu Asp Met Thr Ser Glu Ala Ala Leu Ala LysLeu Ser Tyr Val Leu 325 330 335 Gly Leu Pro Glu Leu Ser Leu Glu Arg ArgGln Glu Leu Leu Ala Lys 340 345 350 Asp Leu Arg Gly Glu Met Thr Leu ProThr Ala Asp Leu His Gln Ser 355 360 365 Ser Pro Pro Gly Ser Thr Leu GlyGln Gly Val Ala Arg Leu Phe Ser 370 375 380 Leu Phe Gly Cys Gln Glu GluAsp Ser Val Gln Asp Ala Val Met Pro 385 390 395 400 Ser Leu Ala Leu AlaLeu Ala His Ala Gly Glu Leu Glu Ala Leu Gln 405 410 415 Ala Leu Met GluLeu Gly Ser Asp Leu Arg Leu Lys Asp Ser Asn Gly 420 425 430 Gln Thr LeuLeu His Val Ala Ala Arg Asn Gly Arg Asp Gly Val Val 435 440 445 Thr MetLeu Leu His Arg Gly Met Asp Val Asn Ala Arg Asp Arg Asp 450 455 460 GlyLeu Ser Pro Leu Leu Leu Ala Val Gln Gly Arg His Arg Glu Cys 465 470 475480 Ile Arg Leu Leu Arg Lys Ala Gly Ala Cys Leu Ser Pro Gln Asp Leu 485490 495 Lys Asp Ala Gly Thr Glu Leu Cys Arg Leu Ala Ser Arg Ala Asp Met500 505 510 Glu Gly Leu Gln Ala Trp Gly Gln Ala Gly Ala Asp Leu Gln GlnPro 515 520 525 Gly Tyr Asp Gly Arg Ser Ala Leu Cys Val Ala Glu Ala AlaGly Asn 530 535 540 Gln Glu Val Leu Ala Leu Leu Arg Asn Leu Ala Leu ValGly Pro Glu 545 550 555 560 Val Pro Pro Ala Ile 565 365 amino acidsamino acid single linear peptide 6 Met Ala Arg Ala Val Gly Pro Glu ArgArg Leu Leu Ala Val Tyr Thr 1 5 10 15 Gly Gly Thr Ile Gly Met Arg SerGlu Leu Gly Val Leu Val Pro Gly 20 25 30 Thr Gly Leu Ala Ala Ile Leu ArgThr Leu Pro Met Phe His Asp Glu 35 40 45 Glu His Ala Arg Ala Arg Gly LeuSer Glu Asp Thr Leu Val Leu Pro 50 55 60 Pro Asp Ser Arg Asn Gln Arg IleLeu Tyr Thr Val Leu Glu Cys Gln 65 70 75 80 Pro Leu Phe Asp Ser Ser AspMet Thr Ile Ala Glu Trp Val Arg Val 85 90 95 Ala Gln Thr Ile Lys Arg HisTyr Glu Gln Tyr His Gly Phe Val Val 100 105 110 Ile His Gly Thr Asp ThrMet Ala Phe Ala Ala Ser Met Leu Ser Phe 115 120 125 Met Leu Glu Asn LeuGln Lys Thr Val Ile Leu Thr Gly Ala Gln Val 130 135 140 Pro Ile His AlaLeu Trp Ser Asp Gly Arg Glu Asn Leu Leu Gly Ala 145 150 155 160 Leu LeuMet Ala Gly Gln Tyr Val Ile Pro Glu Val Cys Leu Phe Phe 165 170 175 GlnAsn Gln Leu Phe Arg Gly Asn Arg Ala Thr Lys Val Asp Ala Arg 180 185 190Arg Phe Ala Ala Phe Cys Ser Pro Asn Leu Leu Pro Leu Ala Thr Val 195 200205 Gly Ala Asp Ile Thr Ile Asn Arg Glu Leu Val Arg Lys Val Asp Gly 210215 220 Lys Ala Gly Leu Val Val His Ser Ser Met Glu Gln Asp Val Gly Leu225 230 235 240 Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ala Leu Val Arg AlaPhe Leu 245 250 255 Gln Pro Pro Leu Lys Gly Val Val Met Glu Thr Phe GlySer Gly Asn 260 265 270 Gly Pro Thr Lys Pro Asp Leu Leu Gln Glu Leu ArgVal Ala Thr Glu 275 280 285 Arg Gly Leu Val Ile Val Asn Cys Thr Gln CysLeu Arg Gly Ala Val 290 295 300 Thr Thr Asp Tyr Ala Ala Gly Met Ala MetAla Gly Ala Asn Val Ile 305 310 315 320 Ser Gly Phe Asp Met Thr Ser GluAla Ala Leu Ala Lys Leu Ser Tyr 325 330 335 Val Leu Gly Gln Pro Gly LeuSer Leu Asp Val Arg Lys Glu Leu Leu 340 345 350 Thr Lys Asp Leu Arg GlyGlu Met Thr Pro Pro Ser Val 355 360 365 365 amino acids amino acidsingle linear peptide 7 Met Ala Arg Ala Val Gly Pro Glu Arg Arg Leu LeuAla Val Tyr Thr 1 5 10 15 Gly Gly Thr Ile Gly Met Arg Ser Glu Leu GlyVal Leu Val Pro Gly 20 25 30 Thr Gly Leu Ala Ala Ile Leu Arg Thr Leu ProMet Phe His Asp Glu 35 40 45 Glu His Ala Arg Ala Arg Gly Leu Ser Glu AspThr Leu Val Leu Pro 50 55 60 Pro Asp Ser Arg Asn Gln Arg Ile Leu Tyr ThrVal Leu Glu Cys Gln 65 70 75 80 Pro Leu Phe Asp Ser Ser Asp Met Thr IleAla Glu Trp Val Arg Val 85 90 95 Ala Gln Thr Ile Lys Arg His Tyr Glu GlnTyr His Gly Phe Val Val 100 105 110 Ile His Gly Thr Asp Thr Met Ala PheAla Ala Ser Met Leu Ser Phe 115 120 125 Met Leu Glu Asn Leu Gln Lys ThrVal Ile Leu Thr Gly Ala Gln Val 130 135 140 Pro Ile His Ala Leu Trp SerAsp Gly Arg Glu Asn Leu Leu Gly Ala 145 150 155 160 Leu Leu Met Ala GlyGln Tyr Val Ile Pro Glu Val Cys Leu Phe Phe 165 170 175 Gln Asn Gln LeuPhe Arg Gly Asn Arg Ala Thr Lys Val Asp Ala Arg 180 185 190 Arg Phe AlaAla Phe Cys Ser Pro Asn Leu Leu Pro Leu Ala Thr Val 195 200 205 Gly AlaAsp Ile Thr Ile Asn Arg Glu Leu Val Arg Lys Val Asp Gly 210 215 220 LysAla Gly Leu Val Val His Ser Ser Met Glu Gln Asp Val Gly Leu 225 230 235240 Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ala Leu Val Arg Ala Phe Leu 245250 255 Gln Pro Pro Leu Lys Gly Val Val Met Glu Thr Phe Gly Ser Gly Asn260 265 270 Gly Pro Thr Lys Pro Asp Leu Leu Gln Glu Leu Arg Val Ala ThrGlu 275 280 285 Arg Gly Leu Val Ile Val Asn Cys Thr Gln Cys Leu Arg GlyAla Val 290 295 300 Thr Thr Asp Tyr Ala Ala Gly Met Ala Met Ala Gly AlaGly Val Ile 305 310 315 320 Ser Gly Phe Asp Met Thr Ser Glu Ala Ala LeuAla Lys Leu Ser Tyr 325 330 335 Val Leu Gly Gln Pro Gly Leu Ser Leu AspVal Arg Lys Glu Leu Leu 340 345 350 Thr Lys Asp Leu Arg Gly Glu Met ThrPro Pro Ser Val 355 360 365 365 amino acids amino acid single linearpeptide 8 Met Ala Arg Ala Val Gly Pro Glu Arg Arg Leu Leu Ala Val TyrThr 1 5 10 15 Gly Gly Thr Ile Gly Met Arg Ser Glu Leu Gly Val Leu ValPro Gly 20 25 30 Thr Gly Leu Ala Ala Ile Leu Arg Thr Leu Pro Met Phe HisAsp Glu 35 40 45 Glu His Ala Arg Ala Arg Gly Leu Ser Glu Asp Thr Leu ValLeu Pro 50 55 60 Pro Asp Ser Arg Asn Gln Arg Ile Leu Tyr Thr Val Leu GluCys Gln 65 70 75 80 Pro Leu Phe Asp Ser Ser Asp Met Thr Ile Ala Glu TrpVal Arg Val 85 90 95 Ala Gln Thr Ile Lys Arg His Tyr Glu Gln Tyr His GlyPhe Val Val 100 105 110 Ile His Gly Thr Asp Thr Met Ala Phe Ala Ala SerMet Leu Ser Phe 115 120 125 Met Leu Glu Asn Leu Gln Lys Thr Val Ile LeuThr Gly Ala Gln Val 130 135 140 Pro Ile His Ala Leu Trp Ser Asp Gly ArgGlu Asn Leu Leu Gly Ala 145 150 155 160 Leu Leu Met Ala Gly Gln Tyr ValIle Pro Glu Val Cys Leu Phe Phe 165 170 175 Gln Asn Gln Leu Phe Arg GlyAsn Arg Ala Thr Lys Val Asp Ala Arg 180 185 190 Arg Phe Ala Ala Phe CysSer Pro Asn Leu Leu Pro Leu Ala Thr Val 195 200 205 Gly Ala Asp Ile ThrIle Asn Arg Glu Leu Val Arg Lys Val Asp Gly 210 215 220 Lys Ala Gly LeuVal Val His Ser Ser Met Glu Gln Asp Val Gly Leu 225 230 235 240 Leu ArgLeu Tyr Pro Gly Ile Pro Ala Ala Leu Val Arg Ala Phe Leu 245 250 255 GlnPro Pro Leu Lys Gly Val Val Met Glu Thr Phe Gly Ser Gly Asn 260 265 270Gly Pro Thr Lys Pro Asp Leu Leu Gln Glu Leu Arg Val Ala Thr Glu 275 280285 Arg Gly Leu Val Ile Val Asn Cys Thr Gln Cys Leu Gln Gly Ala Val 290295 300 Thr Thr Asp Tyr Ala Ala Gly Met Ala Met Ala Gly Ala Asn Val Ile305 310 315 320 Ser Gly Phe Asp Met Thr Ser Glu Ala Ala Leu Ala Lys LeuSer Tyr 325 330 335 Val Leu Gly Gln Pro Gly Leu Ser Leu Asp Val Arg LysGlu Leu Leu 340 345 350 Thr Lys Asp Leu Arg Gly Glu Met Thr Pro Pro SerVal 355 360 365 365 amino acids amino acid single linear peptide 9 MetAla Arg Ala Val Gly Pro Glu Arg Arg Leu Leu Ala Val Tyr Thr 1 5 10 15Gly Gly Thr Ile Gly Met Arg Ser Glu Leu Gly Val Leu Val Pro Gly 20 25 30Thr Gly Leu Ala Ala Ile Leu Arg Thr Leu Pro Met Phe His Asp Glu 35 40 45Glu His Ala Arg Ala Arg Gly Leu Ser Glu Asp Thr Leu Val Leu Pro 50 55 60Pro Asp Ser Arg Asn Gln Arg Ile Leu Tyr Thr Val Leu Glu Cys Gln 65 70 7580 Pro Leu Phe Asp Ser Ser Asp Met Thr Ile Ala Glu Trp Val Arg Val 85 9095 Ala Gln Thr Ile Lys Arg His Tyr Glu Gln Tyr His Gly Phe Val Val 100105 110 Ile His Gly Thr Asp Thr Met Ala Phe Ala Ala Ser Met Leu Ser Phe115 120 125 Met Leu Glu Asn Leu Gln Lys Thr Val Ile Leu Thr Gly Ala GlnVal 130 135 140 Pro Ile His Ala Leu Trp Ser Asp Gly Arg Glu Asn Leu LeuGly Ala 145 150 155 160 Leu Leu Met Ala Gly Gln Tyr Val Ile Pro Glu ValCys Leu Phe Phe 165 170 175 Gln Asn Gln Leu Phe Arg Gly Asn Arg Ala ThrLys Val Asp Ala Arg 180 185 190 Arg Phe Ala Ala Phe Cys Ser Pro Asn LeuLeu Pro Leu Ala Thr Val 195 200 205 Gly Ala Asp Ile Thr Ile Asn Arg GluLeu Val Arg Lys Val Asp Gly 210 215 220 Lys Ala Gly Leu Val Val His SerSer Met Glu Gln Asp Val Gly Leu 225 230 235 240 Leu Arg Leu Tyr Pro GlyIle Pro Ala Ala Leu Val Arg Ala Phe Leu 245 250 255 Gln Pro Pro Leu LysGly Val Val Met Glu Thr Phe Gly Ser Gly Asn 260 265 270 Gly Pro Thr LysPro Asp Leu Leu Gln Glu Leu Arg Val Ala Thr Glu 275 280 285 Arg Gly LeuVal Ile Val Asn Cys Thr Gln Cys Leu Gln Gly Ala Val 290 295 300 Thr ThrAsp Tyr Ala Ala Gly Met Ala Met Ala Gly Ala Gly Val Ile 305 310 315 320Ser Gly Phe Asp Met Thr Ser Glu Ala Ala Leu Ala Lys Leu Ser Tyr 325 330335 Val Leu Gly Gln Pro Gly Leu Ser Leu Asp Val Arg Lys Glu Leu Leu 340345 350 Thr Lys Asp Leu Arg Gly Glu Met Thr Pro Pro Ser Val 355 360 3651089 base pairs nucleic acid single linear 10 ATGGCGCGCG CATCAGGCTCCGAGAGGCAC CTGCTGCTCA TCTACACTGG CGGCACTTTG 60 GGCATGCAGA GCAAGGGCGGGGTGCTCGTC CCCGGCCCAG GCCTGGTCAC TCTGCTGCGG 120 ACCCTGCCCA TGTTCCATGACAAGGAGTTC GCCCAGGCCC AGGGCCTCCC TGACCATGCT 180 CTGGCGCTGC CCCCTGCCAGCCACGGCCCC AGGGTCCTCT ACACGGTGCT GGAGTGCCAG 240 CCCCTCTTGG ATTCCAGCGACATGACCATC GATGATTGGA TTCGCATAGC CAAGATCATA 300 GAGAGGCACT ATGAGCAGTACCAAGGCTTT GTGGTTATCC ACGGCACCGA CACCATGGCC 360 TTTGGGGCCT CCATGCTGTCCTTCATGCTG GAAAACCTGC ACAAACCAGT CATCCTCACT 420 GGCGCCCAGG TGCCAATCCGTGTGCTGTGG AATGACGCCC GGGAAAACCT GCTGGGGGCG 480 TTGCTTGTGG CCGGCCAATACATCATCCCT GAGGTCTGCC TGTTTATGAA CAGTCAGCTG 540 TTTCGGGGAA ACCGGGTAACCAAGGTGGAC TCCCAGAAGT TTGAGGCCTT CTGCTCCCCC 600 AATCTGTCCC CACTAGCCACTGTGGGCGCG GATGTCACAA TTGCCTGGGA CCTGGTGCGC 660 AAGGTCAACT GGAAGGACCCGCTGGTGGTG CACAGCAACA TGGAGCACGA CGTGGCACTG 720 CTGCGCCTCT ACCCTGGCATCCCGGCCTCC CTGGTCCGGG CATTCCTGCA GCCCCCGCTC 780 AAGGGCGTGG TCCTGGAGACCTTCGGCTCT GGCAACGGGC CGAGCAAGCC CGACCTGCTG 840 CAGGAGTTGC GGGCCGCGGCCCAGCGCGGC CTCATCATGG TCAACTGCAG CCAGTGCCTG 900 CGGGGGTCTG TGACCCCGGGCTATGCCACG AGCTTGGCGG GCGCCAACAT CGTGTCCGGC 960 TTAGACATGA CCTCAGAGGCCGCGCTGGCT AAGCTGTCCT ACGTGTTGGG CCTGCCGGAG 1020 CTGAGCCTGG AGCGCAGGCAGGAGCTGCTG GCCAAGGATC TTCGCGGGGA AATGACACTG 1080 CCCACGGCA 1089 1095base pairs nucleic acid single linear 11 ATGGCGCGCG CGGTGGGGCCCGAGCGGAGG CTGCTGGCCG TCTACACCGG CGGCACCATT 60 GGCATGCGGA GTGAGCTCGGCGTGCTTGTG CCCGGGACGG GCCTGGCTGC CATCCTGAGG 120 ACACTGCCCA TGTTCCATGACGAGGAGCAC GCCCGAGCCC GCGGCCTCTC TGAGGACACC 180 CTGGTGCTAC CCCCGGACAGCCGCAACCAG AGGATCCTCT ACACCGTGCT GGAGTGCCAG 240 CCCCTCTTCG ACTCCAGTGACATGACCATC GCTGAGTGGG TTCGCGTTGC CCAGACCATC 300 AAGAGGCACT ACGAGCAGTACCACGGCTTT GTGGTCATCC ACGGCACCGA CACCATGGCC 360 TTTGCTGCCT CGATGCTGTCCTTCATGCTG GAGAACCTGC AGAAGACTGT CATCCTCACT 420 GGGGCCCAGG TGCCCATCCATGCCCTGTGG AGCGACGGCC GTGAGAACCT GCTGGGGGCA 480 CTGCTCATGG CTGGCCAGTATGTGATCCCA GAGGTCTGCC TTTTCTTCCA GAATCAGCTG 540 TTTCGGGGCA ACCGGGCAACCAAGGTAGAC GCTCGGAGGT TCGCAGCTTT CTGCTCCCCG 600 AACCTGCTGC CTCTGGCCACAGTGGGTGCT GACATCACAA TCAACAGGGA GCTGGTGCGG 660 AAGGTGGACG GGAAGGCTGGGCTGGTGGTG CACAGCAGCA TGGAGCAGGA CGTGGGCCTG 720 CTGCGCCTCT ACCCTGGGATCCCTGCCGCC CTGGTTCGGG CCTTCTTGCA GCCTCCCCTG 780 AAGGGCGTGG TCATGGAGACCTTCGGTTCA GGGAACGGAC CCACCAAGCC CGACCTGCTG 840 CAGGAGCTGC GGGTGGCCACCGAGCGCGGC CTGGTCATCG TCAACTGTAC CCAGTGCCTC 900 CGGGGGGCTG TGACCACAGACTATGCAGCT GGCATGGCCA TGGCGGGAGC CAACGTCATC 960 TCAGGCTTCG ACATGACATCGGAGGCCGCC CTGGCCAAGC TATCGTATGT GCTGGGCCAG 1020 CCAGGGCTGA GCCTGGATGTCAGGAAGGAG CTGCTGACCA AGGACCTTCG GGGGGAGATG 1080 ACGCCACCCT CGGTG 10951095 base pairs nucleic acid single linear 12 ATGGCGCGCG CGGTGGGGCCCGAGCGGAGG CTGCTGGCCG TCTACACCGG CGGCACCATT 60 GGCATGCGGA GTGAGCTCGGCGTGCTTGTG CCCGGGACGG GCCTGGCTGC CATCCTGAGG 120 ACACTGCCCA TGTTCCATGACGAGGAGCAC GCCCGAGCCC GCGGCCTCTC TGAGGACACC 180 CTGGTGCTAC CCCCGGACAGCCGCAACCAG AGGATCCTCT ACACCGTGCT GGAGTGCCAG 240 CCCCTCTTCG ACTCCAGTGACATGACCATC GCTGAGTGGG TTCGCGTTGC CCAGACCATC 300 AAGAGGCACT ACGAGCAGTACCACGGCTTT GTGGTCATCC ACGGCACCGA CACCATGGCC 360 TTTGCTGCCT CGATGCTGTCCTTCATGCTG GAGAACCTGC AGAAGACTGT CATCCTCACT 420 GGGGCCCAGG TGCCCATCCATGCCCTGTGG AGCGACGGCC GTGAGAACCT GCTGGGGGCA 480 CTGCTCATGG CTGGCCAGTATGTGATCCCA GAGGTCTGCC TTTTCTTCCA GAATCAGCTG 540 TTTCGGGGCA ACCGGGCAACCAAGGTAGAC GCTCGGAGGT TCGCAGCTTT CTGCTCCCCG 600 AACCTGCTGC CTCTGGCCACAGTGGGTGCT GACATCACAA TCAACAGGGA GCTGGTGCGG 660 AAGGTGGACG GGAAGGCTGGGCTGGTGGTG CACAGCAGCA TGGAGCAGGA CGTGGGCCTG 720 CTGCGCCTCT ACCCTGGGATCCCTGCCGCC CTGGTTCGGG CCTTCTTGCA GCCTCCCCTG 780 AAGGGCGTGG TCATGGAGACCTTCGGTTCA GGGAACGGAC CCACCAAGCC CGACCTGCTG 840 CAGGAGCTGC GGGTGGCCACCGAGCGCGGC CTGGTCATCG TCAACTGTAC CCAGTGCCTC 900 CGGGGGGCTG TGACCACAGACTATGCAGCT GGCATGGCCA TGGCGGGAGC CGGCGTCATC 960 TCAGGCTTCG ACATGACATCGGAGGCCGCC CTGGCCAAGC TATCGTATGT GCTGGGCCAG 1020 CCAGGGCTGA GCCTGGATGTCAGGAAGGAG CTGCTGACCA AGGACCTTCG GGGGGAGATG 1080 ACGCCACCCT CGGTG 10951095 base pairs nucleic acid single linear 13 ATGGCGCGCG CGGTGGGGCCCGAGCGGAGG CTGCTGGCCG TCTACACCGG CGGCACCATT 60 GGCATGCGGA GTGAGCTCGGCGTGCTTGTG CCCGGGACGG GCCTGGCTGC CATCCTGAGG 120 ACACTGCCCA TGTTCCATGACGAGGAGCAC GCCCGAGCCC GCGGCCTCTC TGAGGACACC 180 CTGGTGCTAC CCCCGGACAGCCGCAACCAG AGGATCCTCT ACACCGTGCT GGAGTGCCAG 240 CCCCTCTTCG ACTCCAGTGACATGACCATC GCTGAGTGGG TTCGCGTTGC CCAGACCATC 300 AAGAGGCACT ACGAGCAGTACCACGGCTTT GTGGTCATCC ACGGCACCGA CACCATGGCC 360 TTTGCTGCCT CGATGCTGTCCTTCATGCTG GAGAACCTGC AGAAGACTGT CATCCTCACT 420 GGGGCCCAGG TGCCCATCCATGCCCTGTGG AGCGACGGCC GTGAGAACCT GCTGGGGGCA 480 CTGCTCATGG CTGGCCAGTATGTGATCCCA GAGGTCTGCC TTTTCTTCCA GAATCAGCTG 540 TTTCGGGGCA ACCGGGCAACCAAGGTAGAC GCTCGGAGGT TCGCAGCTTT CTGCTCCCCG 600 AACCTGCTGC CTCTGGCCACAGTGGGTGCT GACATCACAA TCAACAGGGA GCTGGTGCGG 660 AAGGTGGACG GGAAGGCTGGGCTGGTGGTG CACAGCAGCA TGGAGCAGGA CGTGGGCCTG 720 CTGCGCCTCT ACCCTGGGATCCCTGCCGCC CTGGTTCGGG CCTTCTTGCA GCCTCCCCTG 780 AAGGGCGTGG TCATGGAGACCTTCGGTTCA GGGAACGGAC CCACCAAGCC CGACCTGCTG 840 CAGGAGCTGC GGGTGGCCACCGAGCGCGGC CTGGTCATCG TCAACTGTAC CCAGTGCCTC 900 CAGGGGGCTG TGACCACAGACTATGCAGCT GGCATGGCCA TGGCGGGAGC CAACGTCATC 960 TCAGGCTTCG ACATGACATCGGAGGCCGCC CTGGCCAAGC TATCGTATGT GCTGGGCCAG 1020 CCAGGGCTGA GCCTGGATGTCAGGAAGGAG CTGCTGACCA AGGACCTTCG GGGGGAGATG 1080 ACGCCACCCT CGGTG 10951095 base pairs nucleic acid single linear 14 ATGGCGCGCG CGGTGGGGCCCGAGCGGAGG CTGCTGGCCG TCTACACCGG CGGCACCATT 60 GGCATGCGGA GTGAGCTCGGCGTGCTTGTG CCCGGGACGG GCCTGGCTGC CATCCTGAGG 120 ACACTGCCCA TGTTCCATGACGAGGAGCAC GCCCGAGCCC GCGGCCTCTC TGAGGACACC 180 CTGGTGCTAC CCCCGGACAGCCGCAACCAG AGGATCCTCT ACACCGTGCT GGAGTGCCAG 240 CCCCTCTTCG ACTCCAGTGACATGACCATC GCTGAGTGGG TTCGCGTTGC CCAGACCATC 300 AAGAGGCACT ACGAGCAGTACCACGGCTTT GTGGTCATCC ACGGCACCGA CACCATGGCC 360 TTTGCTGCCT CGATGCTGTCCTTCATGCTG GAGAACCTGC AGAAGACTGT CATCCTCACT 420 GGGGCCCAGG TGCCCATCCATGCCCTGTGG AGCGACGGCC GTGAGAACCT GCTGGGGGCA 480 CTGCTCATGG CTGGCCAGTATGTGATCCCA GAGGTCTGCC TTTTCTTCCA GAATCAGCTG 540 TTTCGGGGCA ACCGGGCAACCAAGGTAGAC GCTCGGAGGT TCGCAGCTTT CTGCTCCCCG 600 AACCTGCTGC CTCTGGCCACAGTGGGTGCT GACATCACAA TCAACAGGGA GCTGGTGCGG 660 AAGGTGGACG GGAAGGCTGGGCTGGTGGTG CACAGCAGCA TGGAGCAGGA CGTGGGCCTG 720 CTGCGCCTCT ACCCTGGGATCCCTGCCGCC CTGGTTCGGG CCTTCTTGCA GCCTCCCCTG 780 AAGGGCGTGG TCATGGAGACCTTCGGTTCA GGGAACGGAC CCACCAAGCC CGACCTGCTG 840 CAGGAGCTGC GGGTGGCCACCGAGCGCGGC CTGGTCATCG TCAACTGTAC CCAGTGCCTC 900 CAGGGGGCTG TGACCACAGACTATGCAGCT GGCATGGCCA TGGCGGGAGC CGGCGTCATC 960 TCAGGCTTCG ACATGACATCGGAGGCCGCC CTGGCCAAGC TATCGTATGT GCTGGGCCAG 1020 CCAGGGCTGA GCCTGGATGTCAGGAAGGAG CTGCTGACCA AGGACCTTCG GGGGGAGATG 1080 ACGCCACCCT CGGTG 10951928 base pairs nucleic acid double linear cDNA to mRNA No No guinea pigliver mat peptide 1..19 S 15 GAGTGGCTTA GCCGCAGGC ATG GCG CGC GCA TCAGGC TCC GAG AGG CAC 49 Met Ala Arg Ala Ser Gly Ser Glu Arg His 1 5 10CTG CTG CTC ATC TAC ACT GGC GGC ACT TTG GGC ATG CAG AGC AAG GGC 97 LeuLeu Leu Ile Tyr Thr Gly Gly Thr Leu Gly Met Gln Ser Lys Gly 15 20 25 GGGGTG CTC GTC CCC GGC CCA GGC CTG GTC ACT CTG CTG CGG ACC CTG 145 Gly ValLeu Val Pro Gly Pro Gly Leu Val Thr Leu Leu Arg Thr Leu 30 35 40 CCC ATGTTC CAT GAC AAG GAG TTC GCC CAG GCC CAG GGC CTC CCT GAC 193 Pro Met PheHis Asp Lys Glu Phe Ala Gln Ala Gln Gly Leu Pro Asp 45 50 55 CAT GCT CTGGCG CTG CCC CCT GCC AGC CAC GGC CCC AGG GTC CTC TAC 241 His Ala Leu AlaLeu Pro Pro Ala Ser His Gly Pro Arg Val Leu Tyr 60 65 70 ACG GTG CTG GAGTGC CAG CCC CTC TTG GAT TCC AGC GAC ATG ACC ATC 289 Thr Val Leu Glu CysGln Pro Leu Leu Asp Ser Ser Asp Met Thr Ile 75 80 85 90 GAT GAT TGG ATTCGC ATA GCC AAG ATC ATA GAG AGG CAC TAT GAG CAG 337 Asp Asp Trp Ile ArgIle Ala Lys Ile Ile Glu Arg His Tyr Glu Gln 95 100 105 TAC CAA GGC TTTGTG GTT ATC CAC GGC ACC GAC ACC ATG GCC TTT GGG 385 Tyr Gln Gly Phe ValVal Ile His Gly Thr Asp Thr Met Ala Phe Gly 110 115 120 GCC TCC ATG CTGTCC TTC ATG CTG GAA AAC CTG CAC AAA CCA GTC ATC 433 Ala Ser Met Leu SerPhe Met Leu Glu Asn Leu His Lys Pro Val Ile 125 130 135 CTC ACT GGC GCCCAG GTG CCA ATC CGT GTG CTG TGG AAT GAC GCC CGG 481 Leu Thr Gly Ala GlnVal Pro Ile Arg Val Leu Trp Asn Asp Ala Arg 140 145 150 GAA AAC CTG CTGGGG GCG TTG CTT GTG GCC GGC CAA TAC ATC ATC CCT 529 Glu Asn Leu Leu GlyAla Leu Leu Val Ala Gly Gln Tyr Ile Ile Pro 155 160 165 170 GAG GTC TGCCTG TTT ATG AAC AGT CAG CTG TTT CGG GGA AAC CGG GTA 577 Glu Val Cys LeuPhe Met Asn Ser Gln Leu Phe Arg Gly Asn Arg Val 175 180 185 ACC AAG GTGGAC TCC CAG AAG TTT GAG GCC TTC TGC TCC CCC AAT CTG 625 Thr Lys Val AspSer Gln Lys Phe Glu Ala Phe Cys Ser Pro Asn Leu 190 195 200 TCC CCA CTAGCC ACT GTG GGC GCG GAT GTC ACA ATT GCC TGG GAC CTG 673 Ser Pro Leu AlaThr Val Gly Ala Asp Val Thr Ile Ala Trp Asp Leu 205 210 215 GTG CGC AAGGTC AAC TGG AAG GAC CCG CTG GTG GTG CAC AGC AAC ATG 721 Val Arg Lys ValAsn Trp Lys Asp Pro Leu Val Val His Ser Asn Met 220 225 230 GAG CAC GACGTG GCA CTG CTG CGC CTC TAC CCT GGC ATC CCG GCC TCC 769 Glu His Asp ValAla Leu Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ser 235 240 245 250 CTG GTCCGG GCA TTC CTG CAG CCC CCG CTC AAG GGC GTG GTC CTG GAG 817 Leu Val ArgAla Phe Leu Gln Pro Pro Leu Lys Gly Val Val Leu Glu 255 260 265 ACC TTCGGC TCT GGC AAC GGG CCG AGC AAG CCC GAC CTG CTG CAG GAG 865 Thr Phe GlySer Gly Asn Gly Pro Ser Lys Pro Asp Leu Leu Gln Glu 270 275 280 TTG CGGGCC GCG GCC CAG CGC GGC CTC ATC ATG GTC AAC TGC AGC CAG 913 Leu Arg AlaAla Ala Gln Arg Gly Leu Ile Met Val Asn Cys Ser Gln 285 290 295 TGC CTGCGG GGG TCT GTG ACC CCG GGC TAT GCC ACG AGC TTG GCG GGC 961 Cys Leu ArgGly Ser Val Thr Pro Gly Tyr Ala Thr Ser Leu Ala Gly 300 305 310 GCC AACATC GTG TCC GGC TTA GAC ATG ACC TCA GAG GCC GCG CTG GCT 1009 Ala Asn IleVal Ser Gly Leu Asp Met Thr Ser Glu Ala Ala Leu Ala 315 320 325 330 AAGCTG TCC TAC GTG TTG GGC CTG CCG GAG CTG AGC CTG GAG CGC AGG 1057 Lys LeuSer Tyr Val Leu Gly Leu Pro Glu Leu Ser Leu Glu Arg Arg 335 340 345 CAGGAG CTG CTG GCC AAG GAT CTT CGC GGG GAA ATG ACA CTG CCC ACG 1105 Gln GluLeu Leu Ala Lys Asp Leu Arg Gly Glu Met Thr Leu Pro Thr 350 355 360 GCAGAC CTG CAC CAG TCC TCT CCG CCG GGC AGC ACA CTG GGG CAA GGT 1153 Ala AspLeu His Gln Ser Ser Pro Pro Gly Ser Thr Leu Gly Gln Gly 365 370 375 GTCGCC CGG CTC TTT AGT CTG TTC GGT TGC CAG GAG GAA GAT TCG GTG 1201 Val AlaArg Leu Phe Ser Leu Phe Gly Cys Gln Glu Glu Asp Ser Val 380 385 390 CAGGAC GCC GTG ATG CCC AGC CTG GCC CTG GCC TTG GCC CAT GCT GGT 1249 Gln AspAla Val Met Pro Ser Leu Ala Leu Ala Leu Ala His Ala Gly 395 400 405 410GAA CTC GAG GCT CTG CAG GCA CTT ATG GAG CTG GGC AGT GAC CTG CGC 1297 GluLeu Glu Ala Leu Gln Ala Leu Met Glu Leu Gly Ser Asp Leu Arg 415 420 425CTA AAG GAC TCT AAT GGC CAA ACC CTG TTG CAT GTG GCT GCT CGG AAT 1345 LeuLys Asp Ser Asn Gly Gln Thr Leu Leu His Val Ala Ala Arg Asn 430 435 440GGG CGT GAT GGC GTG GTC ACC ATG CTG CTG CAC AGA GGC ATG GAT GTC 1393 GlyArg Asp Gly Val Val Thr Met Leu Leu His Arg Gly Met Asp Val 445 450 455AAT GCC CGA GAC CGA GAC GGC CTC AGC CCA CTG CTG TTG GCT GTA CAG 1441 AsnAla Arg Asp Arg Asp Gly Leu Ser Pro Leu Leu Leu Ala Val Gln 460 465 470GGC AGG CAT CGG GAA TGC ATC AGG CTG CTG CGG AAG GCT GGG GCC TGC 1489 GlyArg His Arg Glu Cys Ile Arg Leu Leu Arg Lys Ala Gly Ala Cys 475 480 485490 CTG TCC CCC CAG GAC CTG AAG GAT GCA GGG ACC GAG CTG TGC AGG CTG 1537Leu Ser Pro Gln Asp Leu Lys Asp Ala Gly Thr Glu Leu Cys Arg Leu 495 500505 GCA TCC AGG GCT GAC ATG GAA GGC CTG CAG GCA TGG GGG CAG GCT GGG 1585Ala Ser Arg Ala Asp Met Glu Gly Leu Gln Ala Trp Gly Gln Ala Gly 510 515520 GCC GAC CTG CAG CAG CCG GGC TAT GAT GGG CGC AGC GCT CTG TGT GTC 1633Ala Asp Leu Gln Gln Pro Gly Tyr Asp Gly Arg Ser Ala Leu Cys Val 525 530535 GCA GAA GCA GCC GGG AAC CAG GAG GTG CTG GCC CTT CTG CGG AAC CTG 1681Ala Glu Ala Ala Gly Asn Gln Glu Val Leu Ala Leu Leu Arg Asn Leu 540 545550 GCA CTT GTA GGC CCG GAA GTG CCG CCT GCC ATC TGATCGCCAG CAATCCCGCT1734 Ala Leu Val Gly Pro Glu Val Pro Pro Ala Ile 555 560 565 GTGGTGTGAGCCACTCCGCC ATCTGCTGCT TTGACCCACT CGAGGGACCC TAGCACACGA 1794 CCCCCCAGCAGGATGCACCC CACTACTTAG AGTATACCCC AGGCTGGCTC AGTGACAAGC 1854 TGCAAAGGTCTTTGTTGGCA GAACAGCAAT AAAGTAACTA CAGAGTGGCC AAAAAAAAAA 1914 AAAAAAAAAAAAAA 1928 2096 base pairs nucleic acid double linear cDNA to mRNA No Nohuman liver mat peptide 1..92 S 16 CGCCCCGGGC CTCCTCCGCG CAGTCCCTGAGTCCCGCAGG CCCTGCGTCC CCGCTGCACA 60 CCCCCGTCCA CTCCCGTGGT CCCCGGTCCG GCATG GCG CGC GCG GTG GGG CCC 113 Met Ala Arg Ala Val Gly Pro 1 5 GAG CGGAGG CTG CTG GCC GTC TAC ACC GGC GGC ACC ATT GGC ATG CGG 161 Glu Arg ArgLeu Leu Ala Val Tyr Thr Gly Gly Thr Ile Gly Met Arg 10 15 20 AGT GAG CTCGGC GTG CTT GTG CCC GGG ACG GGC CTG GCT GCC ATC CTG 209 Ser Glu Leu GlyVal Leu Val Pro Gly Thr Gly Leu Ala Ala Ile Leu 25 30 35 AGG ACA CTG CCCATG TTC CAT GAC GAG GAG CAC GCC CGA GCC CGC GGC 257 Arg Thr Leu Pro MetPhe His Asp Glu Glu His Ala Arg Ala Arg Gly 40 45 50 55 CTC TCT GAG GACACC CTG GTG CTA CCC CCG GAC AGC CGC AAC CAG AGG 305 Leu Ser Glu Asp ThrLeu Val Leu Pro Pro Asp Ser Arg Asn Gln Arg 60 65 70 ATC CTC TAC ACC GTGCTG GAG TGC CAG CCC CTC TTC GAC TCC AGT GAC 353 Ile Leu Tyr Thr Val LeuGlu Cys Gln Pro Leu Phe Asp Ser Ser Asp 75 80 85 ATG ACC ATC GCT GAG TGGGTT CGC GTT GCC CAG ACC ATC AAG AGG CAC 401 Met Thr Ile Ala Glu Trp ValArg Val Ala Gln Thr Ile Lys Arg His 90 95 100 TAC GAG CAG TAC CAC GGCTTT GTG GTC ATC CAC GGC ACC GAC ACC ATG 449 Tyr Glu Gln Tyr His Gly PheVal Val Ile His Gly Thr Asp Thr Met 105 110 115 GCC TTT GCT GCC TCG ATGCTG TCC TTC ATG CTG GAG AAC CTG CAG AAG 497 Ala Phe Ala Ala Ser Met LeuSer Phe Met Leu Glu Asn Leu Gln Lys 120 125 130 135 ACT GTC ATC CTC ACTGGG GCC CAG GTG CCC ATC CAT GCC CTG TGG AGC 545 Thr Val Ile Leu Thr GlyAla Gln Val Pro Ile His Ala Leu Trp Ser 140 145 150 GAC GGC CGT GAG AACCTG CTG GGG GCA CTG CTC ATG GCT GGC CAG TAT 593 Asp Gly Arg Glu Asn LeuLeu Gly Ala Leu Leu Met Ala Gly Gln Tyr 155 160 165 GTG ATC CCA GAG GTCTGC CTT TTC TTC CAG AAT CAG CTG TTT CGG GGC 641 Val Ile Pro Glu Val CysLeu Phe Phe Gln Asn Gln Leu Phe Arg Gly 170 175 180 AAC CGG GCA ACC AAGGTA GAC GCT CGG AGG TTC GCA GCT TTC TGC TCC 689 Asn Arg Ala Thr Lys ValAsp Ala Arg Arg Phe Ala Ala Phe Cys Ser 185 190 195 CCG AAC CTG CTG CCTCTG GCC ACA GTG GGT GCT GAC ATC ACA ATC AAC 737 Pro Asn Leu Leu Pro LeuAla Thr Val Gly Ala Asp Ile Thr Ile Asn 200 205 210 215 AGG GAG CTG GTGCGG AAG GTG GAC GGG AAG GCT GGG CTG GTG GTG CAC 785 Arg Glu Leu Val ArgLys Val Asp Gly Lys Ala Gly Leu Val Val His 220 225 230 AGC AGC ATG GAGCAG GAC GTG GGC CTG CTG CGC CTC TAC CCT GGG ATC 833 Ser Ser Met Glu GlnAsp Val Gly Leu Leu Arg Leu Tyr Pro Gly Ile 235 240 245 CCT GCC GCC CTGGTT CGG GCC TTC TTG CAG CCT CCC CTG AAG GGC GTG 881 Pro Ala Ala Leu ValArg Ala Phe Leu Gln Pro Pro Leu Lys Gly Val 250 255 260 GTC ATG GAG ACCTTC GGT TCA GGG AAC GGA CCC ACC AAG CCC GAC CTG 929 Val Met Glu Thr PheGly Ser Gly Asn Gly Pro Thr Lys Pro Asp Leu 265 270 275 CTG CAG GAG CTGCGG GTG GCC ACC GAG CGC GGC CTG GTC ATC GTC AAC 977 Leu Gln Glu Leu ArgVal Ala Thr Glu Arg Gly Leu Val Ile Val Asn 280 285 290 295 TGT ACC CACTGC CTC CAG GGG GCT GTG ACC ACA GAC TAT GCA GCT GGC 1025 Cys Thr His CysLeu Gln Gly Ala Val Thr Thr Asp Tyr Ala Ala Gly 300 305 310 ATG GCC ATGGCG GGA GCC GGC GTC ATC TCA GGC TTC GAC ATG ACA TCG 1073 Met Ala Met AlaGly Ala Gly Val Ile Ser Gly Phe Asp Met Thr Ser 315 320 325 GAG GCC GCCCTG GCC AAG CTA TCG TAT GTG CTG GGC CAG CCA GGG CTG 1121 Glu Ala Ala LeuAla Lys Leu Ser Tyr Val Leu Gly Gln Pro Gly Leu 330 335 340 AGC CTG GATGTC AGG AAG GAG CTG CTG ACC AAG GAC CTT CGG GGG GAG 1169 Ser Leu Asp ValArg Lys Glu Leu Leu Thr Lys Asp Leu Arg Gly Glu 345 350 355 ATG ACG CCACCC TCG GTG GAA GAG CGC CGG CCC TCA CTG CAG GGC AAC 1217 Met Thr Pro ProSer Val Glu Glu Arg Arg Pro Ser Leu Gln Gly Asn 360 365 370 375 ACG CTGGGC GGT GGG GTC TCC TGG CTC CTC AGT CTG AGC GGC AGC CAG 1265 Thr Leu GlyGly Gly Val Ser Trp Leu Leu Ser Leu Ser Gly Ser Gln 380 385 390 GAG GCAGAT GCC CTG CGG AAT GCC CTG GTG CCC AGC CTG GCC TGT GCT 1313 Glu Ala AspAla Leu Arg Asn Ala Leu Val Pro Ser Leu Ala Cys Ala 395 400 405 GCT GCCCAC GCC GGT GAC GTG GAG GCG CTG CAG GCG CTT GTG GAG CTG 1361 Ala Ala HisAla Gly Asp Val Glu Ala Leu Gln Ala Leu Val Glu Leu 410 415 420 GGC AGTGAC CTG GGC CTG GTG GAC TTT AAC GGC CAA ACC CCA CTG CAC 1409 Gly Ser AspLeu Gly Leu Val Asp Phe Asn Gly Gln Thr Pro Leu His 425 430 435 GCG GCCGCC CGG GGA GGC CAC ACA GAG GCA GTC ACC ATG CTG CTG CAG 1457 Ala Ala AlaArg Gly Gly His Thr Glu Ala Val Thr Met Leu Leu Gln 440 445 450 455 AGAGGT GTG GAC GTG AAC ACC CGG GAC ACG GAT GGC TTC AGC CCG CTG 1505 Arg GlyVal Asp Val Asn Thr Arg Asp Thr Asp Gly Phe Ser Pro Leu 460 465 470 CTGCTG GCC GTG CGG GGC AGG CAT CCG GGT GTC ATT GGG TTG CTG CGG 1553 Leu LeuAla Val Arg Gly Arg His Pro Gly Val Ile Gly Leu Leu Arg 475 480 485 GAAGCC GGG GCC TCC CTG TCC ACC CAG GAG CTG GAG GAA GCA GGG ACG 1601 Glu AlaGly Ala Ser Leu Ser Thr Gln Glu Leu Glu Glu Ala Gly Thr 490 495 500 GAGCTG TGC AGG CTG GCA TAC AGG GCC GAC CTC GAA GGC CTG CAG GTG 1649 Glu LeuCys Arg Leu Ala Tyr Arg Ala Asp Leu Glu Gly Leu Gln Val 505 510 515 TGGTGG CAG GCA GGG GCT GAC CTG GGG CAG CCG GGC TAT GAC GGG CAC 1697 Trp TrpGln Ala Gly Ala Asp Leu Gly Gln Pro Gly Tyr Asp Gly His 520 525 530 535AGC GCC CTG CAC GTC GCA GAG GCA GCC GGG AAC CTG GCA GTG GTG GCC 1745 SerAla Leu His Val Ala Glu Ala Ala Gly Asn Leu Ala Val Val Ala 540 545 550TTT CTA CAG AGC CTG GAG GGT GCG GTT GGT GCC CAG GCC CCA TGC CCA 1793 PheLeu Gln Ser Leu Glu Gly Ala Val Gly Ala Gln Ala Pro Cys Pro 555 560 565GAA GTG CTG CCT GGT GTC TAACCTGAAG GCGTCCTGCT GCAGTATAAG 1841 Glu ValLeu Pro Gly Val 570 CCATTCCTTC CTCCCATGAC CTGCTGGAGG GGTCTCAGGCATGACCCCAC TGCTGGGGCT 1901 GCTTCCCAGC CTGCTCTCAT GTAAAGCCTG AAGGCCTTTGTTGGGCAGGA CGGCAATAAA 1961 GTCTCTGACA TCCCCTCACC AGGTCTGTAC AGCCTGGCTCTGAGAGGCTC TGTCTGGGCT 2021 CGGGACTGTG AAAAAAAAAA AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAAAA 2081 AAAAAAAAAA AAAAA 2096 1695 base pairs nucleicacid double linear cDNA to mRNA No No guinea pig liver mat peptide1..1695 17 ATG GCG CGC GCA TCA GGC TCC GAG AGG CAC CTG CTG CTC ATC TACACT 48 Met Ala Arg Ala Ser Gly Ser Glu Arg His Leu Leu Leu Ile Tyr Thr 15 10 15 GGC GGC ACT TTG GGC ATG CAG AGC AAG GGC GGG GTG CTC GTC CCC GGC96 Gly Gly Thr Leu Gly Met Gln Ser Lys Gly Gly Val Leu Val Pro Gly 20 2530 CCA GGC CTG GTC ACT CTG CTG CGG ACC CTG CCC ATG TTC CAT GAC AAG 144Pro Gly Leu Val Thr Leu Leu Arg Thr Leu Pro Met Phe His Asp Lys 35 40 45GAG TTC GCC CAG GCC CAG GGC CTC CCT GAC CAT GCT CTG GCG CTG CCC 192 GluPhe Ala Gln Ala Gln Gly Leu Pro Asp His Ala Leu Ala Leu Pro 50 55 60 CCTGCC AGC CAC GGC CCC AGG GTC CTC TAC ACG GTG CTG GAG TGC CAG 240 Pro AlaSer His Gly Pro Arg Val Leu Tyr Thr Val Leu Glu Cys Gln 65 70 75 80 CCCCTC TTG GAT TCC AGC GAC ATG ACC ATC GAT GAT TGG ATT CGC ATA 288 Pro LeuLeu Asp Ser Ser Asp Met Thr Ile Asp Asp Trp Ile Arg Ile 85 90 95 GCC AAGATC ATA GAG AGG CAC TAT GAG CAG TAC CAA GGC TTT GTG GTT 336 Ala Lys IleIle Glu Arg His Tyr Glu Gln Tyr Gln Gly Phe Val Val 100 105 110 ATC CACGGC ACC GAC ACC ATG GCC TTT GGG GCC TCC ATG CTG TCC TTC 384 Ile His GlyThr Asp Thr Met Ala Phe Gly Ala Ser Met Leu Ser Phe 115 120 125 ATG CTGGAA AAC CTG CAC AAA CCA GTC ATC CTC ACT GGC GCC CAG GTG 432 Met Leu GluAsn Leu His Lys Pro Val Ile Leu Thr Gly Ala Gln Val 130 135 140 CCA ATCCGT GTG CTG TGG AAT GAC GCC CGG GAA AAC CTG CTG GGG GCG 480 Pro Ile ArgVal Leu Trp Asn Asp Ala Arg Glu Asn Leu Leu Gly Ala 145 150 155 160 TTGCTT GTG GCC GGC CAA TAC ATC ATC CCT GAG GTC TGC CTG TTT ATG 528 Leu LeuVal Ala Gly Gln Tyr Ile Ile Pro Glu Val Cys Leu Phe Met 165 170 175 AACAGT CAG CTG TTT CGG GGA AAC CGG GTA ACC AAG GTG GAC TCC CAG 576 Asn SerGln Leu Phe Arg Gly Asn Arg Val Thr Lys Val Asp Ser Gln 180 185 190 AAGTTT GAG GCC TTC TGC TCC CCC AAT CTG TCC CCA CTA GCC ACT GTG 624 Lys PheGlu Ala Phe Cys Ser Pro Asn Leu Ser Pro Leu Ala Thr Val 195 200 205 GGCGCG GAT GTC ACA ATT GCC TGG GAC CTG GTG CGC AAG GTC AAC TGG 672 Gly AlaAsp Val Thr Ile Ala Trp Asp Leu Val Arg Lys Val Asn Trp 210 215 220 AAGGAC CCG CTG GTG GTG CAC AGC AAC ATG GAG CAC GAC GTG GCA CTG 720 Lys AspPro Leu Val Val His Ser Asn Met Glu His Asp Val Ala Leu 225 230 235 240CTG CGC CTC TAC CCT GGC ATC CCG GCC TCC CTG GTC CGG GCA TTC CTG 768 LeuArg Leu Tyr Pro Gly Ile Pro Ala Ser Leu Val Arg Ala Phe Leu 245 250 255CAG CCC CCG CTC AAG GGC GTG GTC CTG GAG ACC TTC GGC TCT GGC AAC 816 GlnPro Pro Leu Lys Gly Val Val Leu Glu Thr Phe Gly Ser Gly Asn 260 265 270GGG CCG AGC AAG CCC GAC CTG CTG CAG GAG TTG CGG GCC GCG GCC CAG 864 GlyPro Ser Lys Pro Asp Leu Leu Gln Glu Leu Arg Ala Ala Ala Gln 275 280 285CGC GGC CTC ATC ATG GTC AAC TGC AGC CAG TGC CTG CGG GGG TCT GTG 912 ArgGly Leu Ile Met Val Asn Cys Ser Gln Cys Leu Arg Gly Ser Val 290 295 300ACC CCG GGC TAT GCC ACG AGC TTG GCG GGC GCC AAC ATC GTG TCC GGC 960 ThrPro Gly Tyr Ala Thr Ser Leu Ala Gly Ala Asn Ile Val Ser Gly 305 310 315320 TTA GAC ATG ACC TCA GAG GCC GCG CTG GCT AAG CTG TCC TAC GTG TTG 1008Leu Asp Met Thr Ser Glu Ala Ala Leu Ala Lys Leu Ser Tyr Val Leu 325 330335 GGC CTG CCG GAG CTG AGC CTG GAG CGC AGG CAG GAG CTG CTG GCC AAG 1056Gly Leu Pro Glu Leu Ser Leu Glu Arg Arg Gln Glu Leu Leu Ala Lys 340 345350 GAT CTT CGC GGG GAA ATG ACA CTG CCC ACG GCA GAC CTG CAC CAG TCC 1104Asp Leu Arg Gly Glu Met Thr Leu Pro Thr Ala Asp Leu His Gln Ser 355 360365 TCT CCG CCG GGC AGC ACA CTG GGG CAA GGT GTC GCC CGG CTC TTT AGT 1152Ser Pro Pro Gly Ser Thr Leu Gly Gln Gly Val Ala Arg Leu Phe Ser 370 375380 CTG TTC GGT TGC CAG GAG GAA GAT TCG GTG CAG GAC GCC GTG ATG CCC 1200Leu Phe Gly Cys Gln Glu Glu Asp Ser Val Gln Asp Ala Val Met Pro 385 390395 400 AGC CTG GCC CTG GCC TTG GCC CAT GCT GGT GAA CTC GAG GCT CTG CAG1248 Ser Leu Ala Leu Ala Leu Ala His Ala Gly Glu Leu Glu Ala Leu Gln 405410 415 GCA CTT ATG GAG CTG GGC AGT GAC CTG CGC CTA AAG GAC TCT AAT GGC1296 Ala Leu Met Glu Leu Gly Ser Asp Leu Arg Leu Lys Asp Ser Asn Gly 420425 430 CAA ACC CTG TTG CAT GTG GCT GCT CGG AAT GGG CGT GAT GGC GTG GTC1344 Gln Thr Leu Leu His Val Ala Ala Arg Asn Gly Arg Asp Gly Val Val 435440 445 ACC ATG CTG CTG CAC AGA GGC ATG GAT GTC AAT GCC CGA GAC CGA GAC1392 Thr Met Leu Leu His Arg Gly Met Asp Val Asn Ala Arg Asp Arg Asp 450455 460 GGC CTC AGC CCA CTG CTG TTG GCT GTA CAG GGC AGG CAT CGG GAA TGC1440 Gly Leu Ser Pro Leu Leu Leu Ala Val Gln Gly Arg His Arg Glu Cys 465470 475 480 ATC AGG CTG CTG CGG AAG GCT GGG GCC TGC CTG TCC CCC CAG GACCTG 1488 Ile Arg Leu Leu Arg Lys Ala Gly Ala Cys Leu Ser Pro Gln Asp Leu485 490 495 AAG GAT GCA GGG ACC GAG CTG TGC AGG CTG GCA TCC AGG GCT GACATG 1536 Lys Asp Ala Gly Thr Glu Leu Cys Arg Leu Ala Ser Arg Ala Asp Met500 505 510 GAA GGC CTG CAG GCA TGG GGG CAG GCT GGG GCC GAC CTG CAG CAGCCG 1584 Glu Gly Leu Gln Ala Trp Gly Gln Ala Gly Ala Asp Leu Gln Gln Pro515 520 525 GGC TAT GAT GGG CGC AGC GCT CTG TGT GTC GCA GAA GCA GCC GGGAAC 1632 Gly Tyr Asp Gly Arg Ser Ala Leu Cys Val Ala Glu Ala Ala Gly Asn530 535 540 CAG GAG GTG CTG GCC CTT CTG CGG AAC CTG GCA CTT GTA GGC CCGGAA 1680 Gln Glu Val Leu Ala Leu Leu Arg Asn Leu Ala Leu Val Gly Pro Glu545 550 555 560 GTG CCG CCT GCC ATC 1695 Val Pro Pro Ala Ile 565 1719base pairs nucleic acid double linear cDNA to mRNA No No human liver matpeptide 1..1719 18 ATG GCG CGC GCG GTG GGG CCC GAG CGG AGG CTG CTG GCCGTC TAC ACC 48 Met Ala Arg Ala Val Gly Pro Glu Arg Arg Leu Leu Ala ValTyr Thr 1 5 10 15 GGC GGC ACC ATT GGC ATG CGG AGT GAG CTC GGC GTG CTTGTG CCC GGG 96 Gly Gly Thr Ile Gly Met Arg Ser Glu Leu Gly Val Leu ValPro Gly 20 25 30 ACG GGC CTG GCT GCC ATC CTG AGG ACA CTG CCC ATG TTC CATGAC GAG 144 Thr Gly Leu Ala Ala Ile Leu Arg Thr Leu Pro Met Phe His AspGlu 35 40 45 GAG CAC GCC CGA GCC CGC GGC CTC TCT GAG GAC ACC CTG GTG CTACCC 192 Glu His Ala Arg Ala Arg Gly Leu Ser Glu Asp Thr Leu Val Leu Pro50 55 60 CCG GAC AGC CGC AAC CAG AGG ATC CTC TAC ACC GTG CTG GAG TGC CAG240 Pro Asp Ser Arg Asn Gln Arg Ile Leu Tyr Thr Val Leu Glu Cys Gln 6570 75 80 CCC CTC TTC GAC TCC AGT GAC ATG ACC ATC GCT GAG TGG GTT CGC GTT288 Pro Leu Phe Asp Ser Ser Asp Met Thr Ile Ala Glu Trp Val Arg Val 8590 95 GCC CAG ACC ATC AAG AGG CAC TAC GAG CAG TAC CAC GGC TTT GTG GTC336 Ala Gln Thr Ile Lys Arg His Tyr Glu Gln Tyr His Gly Phe Val Val 100105 110 ATC CAC GGC ACC GAC ACC ATG GCC TTT GCT GCC TCG ATG CTG TCC TTC384 Ile His Gly Thr Asp Thr Met Ala Phe Ala Ala Ser Met Leu Ser Phe 115120 125 ATG CTG GAG AAC CTG CAG AAG ACT GTC ATC CTC ACT GGG GCC CAG GTG432 Met Leu Glu Asn Leu Gln Lys Thr Val Ile Leu Thr Gly Ala Gln Val 130135 140 CCC ATC CAT GCC CTG TGG AGC GAC GGC CGT GAG AAC CTG CTG GGG GCA480 Pro Ile His Ala Leu Trp Ser Asp Gly Arg Glu Asn Leu Leu Gly Ala 145150 155 160 CTG CTC ATG GCT GGC CAG TAT GTG ATC CCA GAG GTC TGC CTT TTCTTC 528 Leu Leu Met Ala Gly Gln Tyr Val Ile Pro Glu Val Cys Leu Phe Phe165 170 175 CAG AAT CAG CTG TTT CGG GGC AAC CGG GCA ACC AAG GTA GAC GCTCGG 576 Gln Asn Gln Leu Phe Arg Gly Asn Arg Ala Thr Lys Val Asp Ala Arg180 185 190 AGG TTC GCA GCT TTC TGC TCC CCG AAC CTG CTG CCT CTG GCC ACAGTG 624 Arg Phe Ala Ala Phe Cys Ser Pro Asn Leu Leu Pro Leu Ala Thr Val195 200 205 GGT GCT GAC ATC ACA ATC AAC AGG GAG CTG GTG CGG AAG GTG GACGGG 672 Gly Ala Asp Ile Thr Ile Asn Arg Glu Leu Val Arg Lys Val Asp Gly210 215 220 AAG GCT GGG CTG GTG GTG CAC AGC AGC ATG GAG CAG GAC GTG GGCCTG 720 Lys Ala Gly Leu Val Val His Ser Ser Met Glu Gln Asp Val Gly Leu225 230 235 240 CTG CGC CTC TAC CCT GGG ATC CCT GCC GCC CTG GTT CGG GCCTTC TTG 768 Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ala Leu Val Arg Ala PheLeu 245 250 255 CAG CCT CCC CTG AAG GGC GTG GTC ATG GAG ACC TTC GGT TCAGGG AAC 816 Gln Pro Pro Leu Lys Gly Val Val Met Glu Thr Phe Gly Ser GlyAsn 260 265 270 GGA CCC ACC AAG CCC GAC CTG CTG CAG GAG CTG CGG GTG GCCACC GAG 864 Gly Pro Thr Lys Pro Asp Leu Leu Gln Glu Leu Arg Val Ala ThrGlu 275 280 285 CGC GGC CTG GTC ATC GTC AAC TGT ACC CAC TGC CTC CAG GGGGCT GTG 912 Arg Gly Leu Val Ile Val Asn Cys Thr His Cys Leu Gln Gly AlaVal 290 295 300 ACC ACA GAC TAT GCA GCT GGC ATG GCC ATG GCG GGA GCC GGCGTC ATC 960 Thr Thr Asp Tyr Ala Ala Gly Met Ala Met Ala Gly Ala Gly ValIle 305 310 315 320 TCA GGC TTC GAC ATG ACA TCG GAG GCC GCC CTG GCC AAGCTA TCG TAT 1008 Ser Gly Phe Asp Met Thr Ser Glu Ala Ala Leu Ala Lys LeuSer Tyr 325 330 335 GTG CTG GGC CAG CCA GGG CTG AGC CTG GAT GTC AGG AAGGAG CTG CTG 1056 Val Leu Gly Gln Pro Gly Leu Ser Leu Asp Val Arg Lys GluLeu Leu 340 345 350 ACC AAG GAC CTT CGG GGG GAG ATG ACG CCA CCC TCG GTGGAA GAG CGC 1104 Thr Lys Asp Leu Arg Gly Glu Met Thr Pro Pro Ser Val GluGlu Arg 355 360 365 CGG CCC TCA CTG CAG GGC AAC ACG CTG GGC GGT GGG GTCTCC TGG CTC 1152 Arg Pro Ser Leu Gln Gly Asn Thr Leu Gly Gly Gly Val SerTrp Leu 370 375 380 CTC AGT CTG AGC GGC AGC CAG GAG GCA GAT GCC CTG CGGAAT GCC CTG 1200 Leu Ser Leu Ser Gly Ser Gln Glu Ala Asp Ala Leu Arg AsnAla Leu 385 390 395 400 GTG CCC AGC CTG GCC TGT GCT GCT GCC CAC GCC GGTGAC GTG GAG GCG 1248 Val Pro Ser Leu Ala Cys Ala Ala Ala His Ala Gly AspVal Glu Ala 405 410 415 CTG CAG GCG CTT GTG GAG CTG GGC AGT GAC CTG GGCCTG GTG GAC TTT 1296 Leu Gln Ala Leu Val Glu Leu Gly Ser Asp Leu Gly LeuVal Asp Phe 420 425 430 AAC GGC CAA ACC CCA CTG CAC GCG GCC GCC CGG GGAGGC CAC ACA GAG 1344 Asn Gly Gln Thr Pro Leu His Ala Ala Ala Arg Gly GlyHis Thr Glu 435 440 445 GCA GTC ACC ATG CTG CTG CAG AGA GGT GTG GAC GTGAAC ACC CGG GAC 1392 Ala Val Thr Met Leu Leu Gln Arg Gly Val Asp Val AsnThr Arg Asp 450 455 460 ACG GAT GGC TTC AGC CCG CTG CTG CTG GCC GTG CGGGGC AGG CAT CCG 1440 Thr Asp Gly Phe Ser Pro Leu Leu Leu Ala Val Arg GlyArg His Pro 465 470 475 480 GGT GTC ATT GGG TTG CTG CGG GAA GCC GGG GCCTCC CTG TCC ACC CAG 1488 Gly Val Ile Gly Leu Leu Arg Glu Ala Gly Ala SerLeu Ser Thr Gln 485 490 495 GAG CTG GAG GAA GCA GGG ACG GAG CTG TGC AGGCTG GCA TAC AGG GCC 1536 Glu Leu Glu Glu Ala Gly Thr Glu Leu Cys Arg LeuAla Tyr Arg Ala 500 505 510 GAC CTC GAA GGC CTG CAG GTG TGG TGG CAG GCAGGG GCT GAC CTG GGG 1584 Asp Leu Glu Gly Leu Gln Val Trp Trp Gln Ala GlyAla Asp Leu Gly 515 520 525 CAG CCG GGC TAT GAC GGG CAC AGC GCC CTG CACGTC GCA GAG GCA GCC 1632 Gln Pro Gly Tyr Asp Gly His Ser Ala Leu His ValAla Glu Ala Ala 530 535 540 GGG AAC CTG GCA GTG GTG GCC TTT CTA CAG AGCCTG GAG GGT GCG GTT 1680 Gly Asn Leu Ala Val Val Ala Phe Leu Gln Ser LeuGlu Gly Ala Val 545 550 555 560 GGT GCC CAG GCC CCA TGC CCA GAA GTG CTGCCT GGT GTC 1719 Gly Ala Gln Ala Pro Cys Pro Glu Val Leu Pro Gly Val 565570 573 29 base pairs nucleic acid single linear cDNA 19 AATCTCGAGCCACCATGGCG CGCGCATCA 29 31 base pairs nucleic acid single linear cDNA 20CTGCGGCCGC TTATCAGATG GCAGGCGGCA C 31 17 amino acids amino acid singlelinear peptide 21 Gly Ser Gly Asn Gly Pro Thr Lys Pro Asp Leu Leu GlnGlu Leu Arg 1 5 10 15 Cys 29 base pairs nucleic acid single linear cDNA22 AATCTCGAGC CACCATGGCG CGCGCGGTG 29 31 base pairs nucleic acid singlelinear cDNA 23 CTGCGGCCGC TTATCAGACA CCAGGCAGCA C 31 31 base pairsnucleic acid single linear cDNA 24 CTGCGGCCGC TTATCATGCC GTGGGCAGTG T 3131 base pairs nucleic acid single linear cDNA 25 CTGCGGCCGC TTATCAGCCCAACACGTAGG A 31 31 base pairs nucleic acid single linear cDNA 26CTGCGGCCGC TCATTACACC GAGGGTGGCG T 31 18 base pairs nucleic acid singlelinear cDNA 27 CCCCCGGAGG CACTGGGT 18 18 base pairs nucleic acid singlelinear cDNA 28 ACCCAGTGCC TCCGGGGG 18 18 base pairs nucleic acid singlelinear cDNA 29 CCCCTGGAGG CACTGGGT 18 18 base pairs nucleic acid singlelinear cDNA 30 ACCCAGTGCC TCCAGGGG 18 18 base pairs nucleic acid singlelinear cDNA 31 CCCCCGGAGG CAGTGGGT 18 18 base pairs nucleic acid singlelinear cDNA 32 ACCCACTGCC TCCGGGGG 18 18 base pairs nucleic acid singlelinear cDNA 33 GACGTTGGCT CCCGCCAT 18 18 base pairs nucleic acid singlelinear cDNA 34 ATGGCGGGAG CCAACGTC 18 23 base pairs nucleic acid singlelinear cDNA 35 GCGAATTCAT GGCGCGCGCA TCA 23 26 base pairs nucleic acidsingle linear cDNA 36 GCAAGCTTTC AGATGGCAGG CGGCAC 26 33 base pairsnucleic acid single linear cDNA 37 GTGAATTCGG AGGTTCAGAT GGCGCGCGCA TCA33 28 base pairs nucleic acid single linear cDNA 38 CTGCGGCCGCTCAGATGGCA GGCGGCAC 28 31 base pairs nucleic acid single linear cDNA 39TCGAGCCACC ATGAAGTGTT CGTGGGTTAT T 31 30 base pairs nucleic acid singlelinear cDNA 40 TTCTTCCTGA TGGCCGTAGT GACAGGAGTG 30 30 base pairs nucleicacid single linear cDNA 41 AATTCACTCC TGTCACTACG GCCATCAGGA 30 31 basepairs nucleic acid single linear cDNA 42 AGAAAATAAC CCACGAACACTTCATGGTGG C 31 26 base pairs nucleic acid single linear cDNA 43GCAAGCTTTC ATGCCGTGGG CAGTGT 26 23 base pairs nucleic acid single linearcDNA 44 GCGAATTCAT GGCGCGCGCG GTG 23 26 base pairs nucleic acid singlelinear cDNA 45 GCAAGCTTTC ACACCGAGGG TGGCGT 26 27 base pairs nucleicacid single linear cDNA 46 CTGCGGCCGC TCATGCCGTG GGCAGTG 27 34 basepairs nucleic acid single linear cDNA 47 CTGAATTCGG AGGTTCAGATGGCGCGCGCG GGTG 34 27 base pairs nucleic acid single linear cDNA 48CTGCGGCCGC TCACACCGAG GGTGGCG 27 565 amino acids amino acid singlelinear peptide 49 Met Ala Arg Ala Ser Gly Ser Glu Arg His Leu Leu LeuIle Tyr Thr 1 5 10 15 Gly Gly Thr Leu Gly Met Gln Ser Lys Gly Gly ValLeu Val Pro Gly 20 25 30 Pro Gly Leu Val Thr Leu Leu Arg Thr Leu Pro MetPhe His Asp Lys 35 40 45 Glu Phe Ala Gln Ala Gln Gly Leu Pro Asp His AlaLeu Ala Leu Pro 50 55 60 Pro Ala Ser His Gly Pro Arg Val Leu Tyr Thr ValLeu Glu Cys Gln 65 70 75 80 Pro Leu Leu Asp Ser Ser Asp Met Thr Ile AspAsp Trp Ile Arg Ile 85 90 95 Ala Lys Ile Ile Glu Arg His Tyr Glu Gln TyrGln Gly Phe Val Val 100 105 110 Ile His Gly Thr Asp Thr Met Ala Phe GlyAla Ser Met Leu Ser Phe 115 120 125 Met Leu Glu Asn Leu His Lys Pro ValIle Leu Thr Gly Ala Gln Val 130 135 140 Pro Ile Arg Val Leu Trp Asn AspAla Arg Glu Asn Leu Leu Gly Ala 145 150 155 160 Leu Leu Val Ala Gly GlnTyr Ile Ile Pro Glu Val Cys Leu Phe Met 165 170 175 Asn Ser Gln Leu PheArg Gly Asn Arg Val Thr Lys Val Asp Ser Gln 180 185 190 Lys Phe Glu AlaPhe Cys Ser Pro Asn Leu Ser Pro Leu Ala Thr Val 195 200 205 Gly Ala AspVal Thr Ile Ala Trp Asp Leu Val Arg Lys Val Asn Trp 210 215 220 Lys AspPro Leu Val Val His Ser Asn Met Glu His Asp Val Ala Leu 225 230 235 240Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ser Leu Val Arg Ala Phe Leu 245 250255 Gln Pro Pro Leu Lys Gly Val Val Leu Glu Thr Phe Gly Ser Gly Asn 260265 270 Gly Pro Ser Lys Pro Asp Leu Leu Gln Glu Leu Arg Ala Ala Ala Gln275 280 285 Arg Gly Leu Ile Met Val Asn Cys Ser Gln Cys Leu Arg Gly SerVal 290 295 300 Thr Pro Gly Tyr Ala Thr Ser Leu Ala Gly Ala Asn Ile ValSer Gly 305 310 315 320 Leu Asp Met Thr Ser Glu Ala Ala Leu Ala Lys LeuSer Tyr Val Leu 325 330 335 Gly Leu Pro Glu Leu Ser Leu Glu Arg Arg GlnGlu Leu Leu Ala Lys 340 345 350 Asp Leu Arg Gly Glu Met Thr Leu Pro ThrAla Asp Leu His Gln Ser 355 360 365 Ser Pro Pro Gly Ser Thr Leu Gly GlnGly Val Ala Arg Leu Phe Ser 370 375 380 Leu Phe Gly Cys Gln Glu Glu AspSer Val Gln Asp Ala Val Met Pro 385 390 395 400 Ser Leu Ala Leu Ala LeuAla His Ala Gly Glu Leu Glu Ala Leu Gln 405 410 415 Ala Leu Met Glu LeuGly Ser Asp Leu Arg Leu Lys Asp Ser Asn Gly 420 425 430 Gln Thr Leu LeuHis Val Ala Ala Arg Asn Gly Arg Asp Gly Val Val 435 440 445 Thr Met LeuLeu His Arg Gly Met Asp Val Asn Ala Arg Asp Arg Asp 450 455 460 Gly LeuSer Pro Leu Leu Leu Ala Val Gln Gly Arg His Arg Glu Cys 465 470 475 480Ile Arg Leu Leu Arg Lys Ala Gly Ala Cys Leu Ser Pro Gln Asp Leu 485 490495 Lys Asp Ala Gly Thr Glu Leu Cys Arg Leu Ala Ser Arg Ala Asp Met 500505 510 Glu Gly Leu Gln Ala Trp Gly Gln Ala Gly Ala Asp Leu Gln Gln Pro515 520 525 Gly Tyr Asp Gly Arg Ser Ala Leu Cys Val Ala Glu Ala Ala GlyAsn 530 535 540 Gln Glu Val Leu Ala Leu Leu Arg Asn Leu Ala Leu Val GlyPro Glu 545 550 555 560 Val Pro Pro Ala Ile 565 573 amino acids aminoacid single linear peptide 50 Met Ala Arg Ala Val Gly Pro Glu Arg ArgLeu Leu Ala Val Tyr Thr 1 5 10 15 Gly Gly Thr Ile Gly Met Arg Ser GluLeu Gly Val Leu Val Pro Gly 20 25 30 Thr Gly Leu Ala Ala Ile Leu Arg ThrLeu Pro Met Phe His Asp Glu 35 40 45 Glu His Ala Arg Ala Arg Gly Leu SerGlu Asp Thr Leu Val Leu Pro 50 55 60 Pro Asp Ser Arg Asn Gln Arg Ile LeuTyr Thr Val Leu Glu Cys Gln 65 70 75 80 Pro Leu Phe Asp Ser Ser Asp MetThr Ile Ala Glu Trp Val Arg Val 85 90 95 Ala Gln Thr Ile Lys Arg His TyrGlu Gln Tyr His Gly Phe Val Val 100 105 110 Ile His Gly Thr Asp Thr MetAla Phe Ala Ala Ser Met Leu Ser Phe 115 120 125 Met Leu Glu Asn Leu GlnLys Thr Val Ile Leu Thr Gly Ala Gln Val 130 135 140 Pro Ile His Ala LeuTrp Ser Asp Gly Arg Glu Asn Leu Leu Gly Ala 145 150 155 160 Leu Leu MetAla Gly Gln Tyr Val Ile Pro Glu Val Cys Leu Phe Phe 165 170 175 Gln AsnGln Leu Phe Arg Gly Asn Arg Ala Thr Lys Val Asp Ala Arg 180 185 190 ArgPhe Ala Ala Phe Cys Ser Pro Asn Leu Leu Pro Leu Ala Thr Val 195 200 205Gly Ala Asp Ile Thr Ile Asn Arg Glu Leu Val Arg Lys Val Asp Gly 210 215220 Lys Ala Gly Leu Val Val His Ser Ser Met Glu Gln Asp Val Gly Leu 225230 235 240 Leu Arg Leu Tyr Pro Gly Ile Pro Ala Ala Leu Val Arg Ala PheLeu 245 250 255 Gln Pro Pro Leu Lys Gly Val Val Met Glu Thr Phe Gly SerGly Asn 260 265 270 Gly Pro Thr Lys Pro Asp Leu Leu Gln Glu Leu Arg ValAla Thr Glu 275 280 285 Arg Gly Leu Val Ile Val Asn Cys Thr His Cys LeuGln Gly Ala Val 290 295 300 Thr Thr Asp Tyr Ala Ala Gly Met Ala Met AlaGly Ala Gly Val Ile 305 310 315 320 Ser Gly Phe Asp Met Thr Ser Glu AlaAla Leu Ala Lys Leu Ser Tyr 325 330 335 Val Leu Gly Gln Pro Gly Leu SerLeu Asp Val Arg Lys Glu Leu Leu 340 345 350 Thr Lys Asp Leu Arg Gly GluMet Thr Pro Pro Ser Val Glu Glu Arg 355 360 365 Arg Pro Ser Leu Gln GlyAsn Thr Leu Gly Gly Gly Val Ser Trp Leu 370 375 380 Leu Ser Leu Ser GlySer Gln Glu Ala Asp Ala Leu Arg Asn Ala Leu 385 390 395 400 Val Pro SerLeu Ala Cys Ala Ala Ala His Ala Gly Asp Val Glu Ala 405 410 415 Leu GlnAla Leu Val Glu Leu Gly Ser Asp Leu Gly Leu Val Asp Phe 420 425 430 AsnGly Gln Thr Pro Leu His Ala Ala Ala Arg Gly Gly His Thr Glu 435 440 445Ala Val Thr Met Leu Leu Gln Arg Gly Val Asp Val Asn Thr Arg Asp 450 455460 Thr Asp Gly Phe Ser Pro Leu Leu Leu Ala Val Arg Gly Arg His Pro 465470 475 480 Gly Val Ile Gly Leu Leu Arg Glu Ala Gly Ala Ser Leu Ser ThrGln 485 490 495 Glu Leu Glu Glu Ala Gly Thr Glu Leu Cys Arg Leu Ala TyrArg Ala 500 505 510 Asp Leu Glu Gly Leu Gln Val Trp Trp Gln Ala Gly AlaAsp Leu Gly 515 520 525 Gln Pro Gly Tyr Asp Gly His Ser Ala Leu His ValAla Glu Ala Ala 530 535 540 Gly Asn Leu Ala Val Val Ala Phe Leu Gln SerLeu Glu Gly Ala Val 545 550 555 560 Gly Ala Gln Ala Pro Cys Pro Glu ValLeu Pro Gly Val 565 570 573

What is claimed is:
 1. A purified polypeptide capable of exhibitingL-asparaginase activity, consisting of either an amino acid sequenceconsisting of a part of SEQ ID NO:9 with the remainder of SEQ ID NO:9substituted with the corresponding part oft SEQ ID NO:4, or a fragmentof said amino acid sequencer which contains a part of part SEQ ID NO:9and a part of SEQ ID NO:4, and which is capable of exhibitingL-asparaginase activity.
 2. The purified polypeptide of claim 1, whoseenzymatically active form is an oligomeric form.
 3. A pharmaceuticalcomposition, comprising the purified polypeptide of claim 1 as aneffective ingredient and a pharmaceutically-acceptable diluent,excipient, carrier or auxiliary agent.
 4. The pharmaceutical compositionaccording to claim 3, which is used to treat malignant tumors, leukemiaand lymphomas.
 5. The pharmaceutical composition according to claim 3,further comprising one or more stabilizers selected from the groupconsisting of serum albumin, glycerol, gelatin, trehalose, and maltose.6. A purified polypeptide f agment capable of exhibiting L-asparaginaseactivity, wherein said polypeptide fragment is a fragment of thepolypeptide of SEQ ID NO:49 and comprises the amino acid sequence of SEQID NO:4.
 7. The purified polypeptide fragment of claim 6, whoseenzymatically active form is an oligomeric form.
 8. A pharmaceuticalcomposition, comprising the purified polypeptide fragment of claim 6 asan effective ingredient and a pharmaceutically-acceptable diluent,excipient, carrier or auxiliary agent.
 9. The pharmaceutical compositionaccording to claim 8, which is used to treat malignant tumors, leukemiaand lymphomas.
 10. The pharmaceutical composition according to claim 8,further comprising one or more stabilizers selected from the groupconsisting of serum albumin, glycerol, gelatin, trehalose, and maltose.